Biomarker for cognitive dysfunction diseases, and method for detection of cognitive dysfunction diseases using the biomarker

ABSTRACT

The present invention aims to provide methods to detect cognitive impairment including mild cognitive impairment and Alzheimer disease by using a protein or its partial peptide that differs in presence or absence, or in quantity between non-cognitive impairment and patients with cognitive impairment and further aims to present biomarkers comprising said protein and said partial peptide to be used to detect cognitive impairment including Alzheimer disease or mild cognitive impairment. Specifically, a biomarker for diagnosis of psychiatry disease or cognitive impairment comprising protein fragment or peptide of not less than 5 amino acid residues arising from at least one protein or peptide selected from the group of proteins consisting of amino acid sequence expressed by SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, and 25 and selected from the group of partial peptide in these proteins consisting of amino acid sequence expressed by SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 27. And further aims to provide diagnostic method using these biomarker.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of copending application Ser. No.15/467,646, filed on Mar. 23, 2017, which is a Continuation of Ser. No.14/582,778, filed on Dec. 24, 2014, which is a Continuation ofapplication Ser. No. 13/995,682, filed on Sep. 3, 2013 (now abandoned),which was filed as PCT International Application No. PCT/JP2011/007150on Dec. 21, 2011, which claims the benefit under 35 U.S.C. §119(a) toPatent Application No. 2010-285726, filed in JAPAN on Dec. 22, 2010, allof which are hereby expressly incorporated by reference into the presentapplication.

FIELD OF THE INVENTION

The present invention relates to novel biomarkers for mild cognitiveimpairment or cognitive impairment including Alzheimer disease, andmethods for detecting cognitive impairment using such biomarkers.

BACKGROUND OF THE INVENTION

The commonly used means to differentiate between normal and non-normalstates of a human subject using his or her biological materials aremainly those which have been used in the field of diagnostics. Mostfrequently used are those methods which target biomarkers in blood. Ithas been practiced in this field to comparatively measure the amount ofa specific protein or a peptide that is less than 10,000 in molecularweight or, in the case of enzyme protein, enzyme activities in samplesfrom normal (healthy) subjects and those from diseased individuals tohelp diagnosis. Here, prior to testing real samples, measurements aredone on a fixed number each of samples from healthy controls andpatients with certain disease with respect to the amount (s) or activity(activities) of single or multiple specific proteins or peptides and theranges of abnormal and normal values are respectively determined. Thesample to be evaluated is then analyzed by the same method and theresultant value is judged with respect to whether it is in normal orabnormal range.

In the actual measurements, the amount(s) of specified protein(s) orpeptide(s) in test samples, as such or after dilution, are determined bythe use of enzyme-linked immmunosorbent assay (ELISA) which uses aprimary, or secondary, antibody labeled with an enzyme reacting with asubstrate that yields a color upon reaction, chemiluminescentimmunoassay (CLIA), radioimmunoassay (RIA) which uses a primary, orsecondary, antibody labeled with a radioisotope, and, if the protein isan enzyme, the measurement of the activity of the enzyme by adding itssubstrate and determining the intensity of produced color, etc. Theseantibody-based methods are called as enzyme-, fluorescence- orradioisotope-labeled methods, respectively. In addition, there is amethod where an enzyme reaction product derived from the correspondingsubstrate is determined by high performance liquid chromatography(HPLC). In further addition, there is a method where HPLC is combinedwith mass spectrometer, called LC-MS/MS, and there is a method calledselected reaction monitoring (SRM)/multiple reaction monitoring (MRM)that utilizes LC-MS/MS. In another method to determine the concentrationin a sample, it is appropriately pretreated, and separation of proteinsor peptides is attained by 2-dimensional polyacrylamide gelelectrophoresis (2D-PAGE), and target protein or peptide is determinedby silver staining, Coomassie blue staining or immunological staining(Western blotting) that uses an antibody to target protein or peptide.In still further addition, there is a method which utilizes massspectrometry to determine the amount of target protein or peptide insamples fractionated by column chromatography. Instead of columnchromatography, protein chips and magnetic beads may also be utilizedfor purpose of pretreatment.

Furthermore, these inventors have developed an immunoMS method, wheretarget protein or peptide is captured by beads (including magnetic ones)with linked antibody to the protein or peptide, eluted from the beads,and determined by mass spectrometry. Further, intact proteins have beenreported to be analyzed by mass spectrometry using above-mentionedmethods after digestion with trypsin etc. (Patent Document 1). Here,intact target proteins are selected either by fractionation or byadsorption to an adsorbant specific to them and then determined by massspectrometry.

Number of patients suffered from cognitive impairment like Alzheimerdisease is increasing rapidly along with increasing of old-agepopulation in Japan. It is estimated that number of patients is 1.3million in 1995 and it will be 1.9 million in 2005 and will reach toabout 3.0 million in 2020. It is reported that 60-90% of cognitiveimpairment is Alzheimer disease. As manifestation of Alzheimer diseaseis not only loss of memory but several disturbance in daily life,increase of patients of this disease is becoming an important socialissue to be solved. In Japan, Donepezil-hydrochloride,anti-acetylcholine esterase inhibitor has been available for medicaltreatment for Alzheimer disease since 1999, and it let progress ofcognitive impairment in these patients be ‘slow-down’ efficiently, ifthe patient is diagnosed at early stage. Thus, in medication ofAlzheimer disease, most important issue is ‘early diagnosis’ to treatthe patients effectively by drug available at present and new comingdrug.

Followings are major criteria for diagnosis of Alzheimer diseasedescribed in DSM-IV, which is published by American PsychiatricAssociation.

-   -   A. The development of multiple cognitive deficits manifested by        both    -   (1) memory impairment (impaired ability to learn new information        or to recall previously learned information)    -   (2) one (or more) of the following cognitive disturbances:        -   a) aphasia (language disturbance)        -   b) apraxia (impaired ability to carry out motor activities            despite intact motor function)        -   c) agnosia (failure to recognize or identify objects despite            intact sensory function)        -   d) disturbance in executive functioning (i.e., planning,            organizing, sequencing, abstracting)    -   B. The cognitive deficits in Criteria A1 and A2 each cause        significant impairment in social or occupational functioning and        represent a significant decline from a previous level of        functioning. (Non-patent reference 1)

There are several types of neurological disorders related to Alzheimerdisease (AD). As cognitive dysfunction appears gradually in dementiaincluding AD, there is a disease status of pre-stage of dementia. Thisstage is called as mild cognitive impairment (MCI). In United States,10% MCI develops to AD within 1 year, and 50% of MCI develops to ADwithin 4 years. MCI is defined as a condition characterized by newlyacquired cognitive decline to an extent that is beyond that expected forage or educational background, yet not causing significant functionalimpairment, and not showing disturbance in daily life. Frontotemporaldementia (frontotemporal lobar degeneration) (FTD) shows loss ofpersonal awareness, loss of social awareness, hyperorality, andstereotyped, perseverative behavior. These clinical characteristics aredifferent from AD. FTD includes Pick's disease, which is characterizedby microscopically Pick bodies usually found in limbic, paralimbic, andventral temporal lobe cortex. Dementia with Lewy bodies (DLB) ischaracterized by progressive disease and psychiatric symptoms includeanxiety, depression, hallucinations (usually visual) and delusions(false beliefs). DLB is thought to be the second most common subtype and10-30% of dementia is DLB. The symptoms of DLB are caused by thebuild-up of Lewy bodies. FTD and DLB belong to demented neurologicaldisease as they also lose of memory, their ability to solve problems andmaintain emotional control. (Non-patent reference 1)

In description in present patent, cognitive impairment includes AD, MCIand the demented neurological disease.

The screening tests for dementia widely used are the Hasegawa DementiaScale-revised (HDS-R) and Mini-Mental State Examination (MMSE). In thesescreening tests, inspector asks several questions and evaluates level ofcognitive impairment of each subject by scores. HDS-R is revised versionof HDS published in 1991. In HDS-R, test consists of 9 questions toanalyses orientation, remembrance, calculation, retain and recallability, and common sense. Full score is 30 and a person whose score isless than 23 is suspected as dementia. MMSE has been developed in UnitedStates to screen and diagnose dementia, and analyses global cognitivefunction, with items assessing orientation, word recall, attention andcalculation, language abilities, and visuospatial (drawing) ability.This test consists of 11 questions, and full score is 30 and a personwho has score less than 23 is suspected as dementia. The results ofHDS-R and MMSE coincide with each other. Both are used for screening,not for diagnosis and not for staging of disease progression.(Non-patent reference 1)

Neuroimaging test for dementia are Computed tomography (CT) and Magneticresonance imaging (MRI) which evaluate morphological changes like brainatrophy and ventricular dilation and single-photon emission computedtomography (SPECT) which analyses regional cerebral blood flow and PETwhich shows brain metabolism by measurement of consumption of oxygen andsugar. SPECT and PET, nuclear imaging technologies, can identifyneuronal dysfunction at preclinical stage. However, these neuroimagingcannot be widely used in hospitals because they need special facilitiesto perform nuclear imaging, and neuroimaging may not be objective testas imaging diagnosis is completely depend on the skill of physician whoanalyses the mages.

Thus, methods for screening and diagnosis of dementia including AD thatare available at present is dependent on tests lacking objectivity andis dependent on expensive instruments, and so it is very difficult touse these tests for screening of early stage-cognitive impairment. If weget blood (serum/plasma) biomarker for cognitive impairment, whichenables us objective test using specimens we can easily obtain, we canidentify cognitive impairment at early stage (preclinical stage) byblood test using such biomarker. Present patent provides novelbiomarkers and a novel and potent diagnostic method for cognitiveimpairment by using such biomarkers and biomarkers described here.

CITATION LIST Patent Document

-   Patent Document 1, JP-A-2004-333274-   Patent Document 2, JP-A-2006-308533

Non-Patent Document

-   Non-Patent Document 1, “The better understanding of Alzheimer's    disease.,” edited by Imaharu Nakano and Hldehiro Mizusawa., Nagai    Shoten Co., Ltd., 2004 (in Japanese)-   Non-Patent Document 2, Benkirane, N. et al., J. Biol. Chem. Vol.    268, 26279-26285, 1993

SUMMARY OF THE INVENTION Technical Problem

The present invention aims to present methods to detect mild cognitiveimpairment or cognitive impairment including Alzheimer disease by usinga protein or its partial peptide that differs in presence or absence, orin quantity between non-cognitive impairment subjects (including healthypeople, the human subjects that may be affected with any disease andunaffected with psychiatry disease including cognitive impairment. Thesehuman subjects are allowed to match the age and gender of patient withcognitive impairment. And, these human subjects are called non-dementedcontrol, hereinafter abbreviated to NDC.) and patients with cognitiveimpairment and further aims to present biomarkers comprising saidproteins and said partial peptides to be used to detect mild cognitiveimpairment or cognitive impairment including Alzheimer disease.

Solution to Problem

These inventors investigated to find out means to detect cognitiveimpairment and found a peptide capable of detecting mild cognitiveimpairment or cognitive impairment including Alzheimer disease in theserum. Said peptides found in the present invention are those withsignificance as a biomarker to detecting in the case of serum not onlyother biological materials such as blood, plasma, cerebrospinal fluid,and urine. Simultaneously, protein or peptide is the origin of thesepeptides (hereinafter referred to as intact proteins or peptides) alsohas significance as biomarkers.

Specifically, these inventors found that a biomarker comprising at leastone protein or peptide selected from the group consisting of ComplementC3 consisting of amino acid sequence expressed by SEQ ID NO: 1,Transcription factor AP-2 gamma consisting of amino acid sequenceexpressed by SEQ ID NO: 3, Synapsin-3 consisting of amino acid sequenceexpressed by SEQ ID NO: 5, Oxytocin receptor consisting of amino acidsequence expressed by SEQ ID NO: 7, Inter-alpha-trypsin inhibitor heavychain H5-like protein consisting of amino acid sequence expressed by SEQID NO: 9, E3 ubiquitin-protein ligase HERC2 consisting of amino acidsequence expressed by SEQ ID NO: 11, Prothrombin consisting of aminoacid sequence expressed by SEQ ID NO: 13, Transthyretin consisting ofamino acid sequence expressed by SEQ ID NO: 15, Tumor necrosis factorreceptor superfamily member 16 consisting of amino acid sequenceexpressed by SEQ ID NO: 17, Complement C4-A consisting of amino acidsequence expressed by SEQ ID NO: 19, Complement C4-B consisting of aminoacid sequence expressed by SEQ ID NO: 21, Fibrinogen alpha chain(isoform 1) consisting of amino acid sequence expressed by-SEQ ID NO:23, and Fibrinogen alpha chain (isoform 2) consisting of amino acidsequence expressed by SEQ ID NO: 25; or a biomarker comprising proteinfragment or peptide of not less than 5 amino acid residues arising fromat least one protein or peptide selected from the group consisting ofthem, could be used as biomarkers to detect cognitive impairment.

Furthermore, these inventors found that a biomarker comprising from thegroup consisting of Complement C3-derived peptide CO3 consisting ofamino acid sequence expressed by SEQ ID NO: 2, Transcription factor AP-2gamma-derived peptide AP2C consisting of amino acid sequence expressedby SEQ ID NO: 4, Synapsin-3-derived peptide SYN3 consisting of aminoacid sequence expressed by SEQ ID NO: 6, Oxytocin receptor-derivedpeptide OXYR consisting of amino acid sequence expressed by SEQ ID NO:8, Inter-alpha-trypsin inhibitor heavy chain H5-like protein-derivedpeptide ITH5L consisting of amino acid sequence expressed by SEQ ID NO:10, E3 ubiquitin-protein ligase HERC2-derived peptide HERC2 consistingof amino acid sequence expressed by SEQ ID NO: 12, Prothrombin-derivedpeptide THRB consisting of amino acid sequence expressed by SEQ ID NO:14, Transthyretin-derived peptide TTHY consisting of amino acid sequenceexpressed by SEQ ID NO: 16, Tumor necrosis factor receptor superfamilymember 16-derived peptide TNR16 consisting of amino acid sequenceexpressed by SEQ ID NO: 18, Complement C4-derived peptide C04-1consisting of amino acid sequence expressed by SEQ ID NO: 20, ComplementC4-derived peptide C04-2 consisting of amino acid sequence expressed bySEQ ID NO: 22, Fibrinogen alpha chain-derived peptide FIBA-1 consistingof amino acid sequence expressed by SEQ ID NO: 24, Fibrinogen alphachain-derived peptide FIBA-2 consisting of amino acid sequence expressedby SEQ ID NO: 26, and Fibrinogen alpha chain-derived peptide FIBA-3consisting of amino acid sequence expressed by SEQ ID NO: 27 could beused as biomarkers to detect cognitive impairment.

These inventors brought the present invention to perfection by furthersucceeding in determining simultaneously these many proteins and itspartial peptides by using two-dimensional high performance liquidchromatography-MALDI TOF-MS method (mass spectrometry) and immunoMSmethod.

The features of the present invention are shown below.

[1] A biomarker for detection of cognitive impairment comprising proteinfragment or peptide of not less than 5 amino acid residues arising fromat least one protein or peptide selected from the group consisting ofComplement C3 consisting of amino acid sequence expressed by SEQ ID NO:1, Transcription factor AP-2 gamma consisting of amino acid sequenceexpressed by SEQ ID NO: 3, Synapsin-3 consisting of amino acid sequenceexpressed by SEQ ID NO: 5, Oxytocin receptor consisting of amino acidsequence expressed by SEQ ID NO: 7, Inter-alpha-trypsin inhibitor heavychain H5-like protein consisting of amino acid sequence expressed by SEQID NO: 9, E3 ubiquitin-protein ligase HERC2 consisting of amino acidsequence expressed by SEQ ID NO: 11, Prothrombin consisting of aminoacid sequence expressed by SEQ ID NO: 13, Transthyretin consisting ofamino acid sequence expressed by SEQ ID NO: 15, Tumor necrosis factorreceptor superfamily member 16 consisting of amino acid sequenceexpressed by SEQ ID NO: 17, Complement C4-A consisting of amino acidsequence expressed by SEQ ID NO: 19, Complement C4-B consisting of aminoacid sequence expressed by SEQ ID NO: 21, Fibrinogen alpha chain(isoform 1) consisting of amino acid sequence expressed by SEQ ID NO:23, and Fibrinogen alpha chain (isoform 2) consisting of amino acidsequence expressed by SEQ ID NO: 25, or a biomarker for detection ofcognitive impairment comprising at least one protein or peptide selectedfrom the group consisting of them.

[2] A biomarker for detection of cognitive impairment comprising thepeptide selected from the group consisting of Complement C3-derivedpeptide CO3 consisting of amino acid sequence expressed by SEQ ID NO: 2,Transcription factor AP-2 gamma-derived peptide AP2C consisting of aminoacid sequence expressed by SEQ ID NO: 4, Synapsin-3-derived peptide SYN3consisting of amino acid sequence expressed by-SEQ ID NO: 6, Oxytocinreceptor-derived peptide OXYR consisting of amino acid sequenceexpressed by SEQ ID NO: 8, Inter-alpha-trypsin inhibitor heavy chainH5-like protein-derived peptide ITH5L consisting of amino acid sequenceexpressed by SEQ ID NO: 10, E3 ubiquitin-protein ligase HERC2-derivedpeptide I-IERC2 consisting of amino acid sequence expressed by SEQ IDNO: 12, Prothrombin-derived peptide THRB consisting of amino acidsequence expressed by SEQ ID NO: 14, Transthyretin-derived peptide TTHYconsisting of amino acid sequence expressed by SEQ ID NO: 16, Tumornecrosis factor receptor superfamily member 16-derived peptide TNR16consisting of amino acid sequence expressed by SEQ ID NO: 18, ComplementC4-derived peptide C04-1 consisting of amino acid sequence expressed bySEQ ID NO: 20, Complement C4-derived peptide C04-2 consisting of aminoacid sequence expressed by SEQ ID NO: 22, Fibrinogen alpha chain-derivedpeptide FIBA-1 consisting of amino acid sequence expressed by SEQ ID NO:24, Fibrinogen alpha chain-derived peptide FIBA-2 consisting of aminoacid sequence expressed by SEQ ID NO: 26, and Fibrinogen alphachain-derived peptide FIBA-3 consisting of amino acid sequence expressedby SEQ ID NO: 27, or a biomarker for detection of cognitive impairmentcomprising at least one protein or peptide selected from the groupconsisting of them.

[3] A biomarker of cognitive impairment comprising the peptides selectedfrom the group consisting of amino acid sequence expressed by SEQ IDNOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 27 that isappeared or increased in biological material of patients of cognitiveimpairment as compared to biological material of subjects not sufferingfrom psychiatry disease.

[4] A biomarker of Alzheimer disease comprising the peptides selectedfrom the group consisting of amino acid sequence expressed by SEQ IDNOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 27 that isappeared or increased in biological material of patients of Alzheimerdisease as compared to biological material of subjects not sufferingfrom psychiatry disease.

[5] A biomarker of mild cognitive impairment comprising the peptidesselected from the group consisting of amino acid sequence expressed bySEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 27 thatis appeared or increased in biological material of patients of mildcognitive impairment as compared to biological material of subjects notsuffering from psychiatry disease.

[6] Method for detection of cognitive impairment involving determinationin biological material of at least one biomarker for cognitiveimpairment described in any of [1] to [5].

[7] Method for detection of psychiatry disease described in [6] whereindetection is made either by immunoblot procedure, Western blotting,enzyme-, fluorescence-, or radioisotope-labeled antibody method, massspectrometry, immunoMS method or surface plasmon resonance method.

[8] A kit for detection of cognitive impairment to determine at leastone biomarker described in any of [1] to [5].

[9] A kit for detection of psychiatry disease containing antibody oraptamer to at least one biomarker described in any of [1] to [5].

Advantageous Effect of the Invention

According to the present invention, it is possible to diagnose thesubject such as suffering from mild cognitive impairment or cognitiveimpairment including Alzheimer's disease, when to increase or appearcompared to the biological sample of subjects not suffering frompsychiatry disease by determining amount of at least one biomarkercomprising protein fragment or peptide of not less than 5 amino acidresidues arising from at least one protein or peptide selected from thegroup consisting of Complement C3 consisting of amino acid sequenceexpressed by SEQ ID NO: 1, Transcription factor AP-2 gamma consisting ofamino acid sequence expressed by SEQ ID NO: 3, Synapsin-3 consisting ofamino acid sequence expressed by SEQ ID NO: 5, Oxytocin receptorconsisting of amino acid sequence expressed by SEQ ID NO: 7,Inter-alpha-trypsin inhibitor heavy chain H5-like protein consisting ofamino acid sequence expressed by SEQ ID NO: 9, E3 ubiquitin-proteinligase HERC2 consisting of amino acid sequence expressed by SEQ ID NO:11, Prothrombin consisting of amino acid sequence expressed by SEQ IDNO: 13, Transthyretin consisting of amino acid sequence expressed by SEQID NO: 15, Tumor necrosis factor receptor superfamily member 16consisting of amino acid sequence expressed by SEQ ID NO: 17, ComplementC4-A consisting of amino acid sequence expressed by SEQ ID NO: 19,Complement C4-B consisting of amino acid sequence expressed by SEQ IDNO: 21, Fibrinogen alpha chain (isoform 1) consisting of amino acidsequence expressed by SEQ ID NO: 23, and Fibrinogen alpha chain (isoform2) consisting of amino acid sequence expressed by SEQ ID NO: 25.

In addition, according to the present invention, it is possible todiagnose the subject such as suffering from mild cognitive impairment orcognitive impairment including Alzheimer's disease, when to increase orappear compared to the biological sample of subjects not suffering frompsychiatry disease by determining kind or amount at least one peptideselected from the group consisting of Complement C3-derived peptide CO3consisting of amino acid sequence expressed by SEQ ID NO: 2,Transcription factor AP-2 gamma-derived peptide AP2C consisting of aminoacid sequence expressed by SEQ ID NO: 4, Synapsin-3-derived peptide SYN3consisting of amino acid sequence expressed by SEQ ID NO: 6, Oxytocinreceptor-derived peptide OXYR consisting of amino acid sequenceexpressed by SEQ ID NO: 8, Inter-alpha-trypsin inhibitor heavy chainH5-like protein-derived peptide ITH5L consisting of amino acid sequenceexpressed by SEQ ID NO: 10, E3 ubiquitin-protein ligase HERC2-derivedpeptide HERC2 consisting of amino acid sequence expressed by SEQ ID NO:12, Prothrombin-derived peptide THRB consisting of amino acid sequenceexpressed by SEQ ID NO: 14, Transthyretin-derived peptide TTHYconsisting of amino acid sequence expressed by SEQ ID NO: 16, Tumornecrosis factor receptor superfamily member 16-derived peptide TNR16consisting of amino acid sequence expressed by SEQ ID NO: 18, ComplementC4-derived peptide C04-1 consisting of amino acid sequence expressed bySEQ ID NO: 20, Complement C4-derived peptide C04-2 consisting of aminoacid sequence expressed by SEQ ID NO: 22, Fibrinogen alpha chain-derivedpeptide FIBA-1 consisting of amino acid sequence expressed by SEQ ID NO:24, Fibrinogen alpha chain-derived peptide FIBA-2 consisting of aminoacid sequence expressed by SEQ ID NO: 26, and Fibrinogen alphachain-derived peptide FIBA-3 consisting of amino acid sequence expressedby SEQ ID NO: 27.

The present invention provides a diagnostic system that is high in bothaccuracy and specificity. The present invention enables highly accuratediagnosis of cognitive impairment in which there have been no specifictest methods for such biological materials as blood. Furthermore, thebiomarkers disclosed in the present invention are highly useful injudgment of drug efficacy.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates the cluster map of Marker A. The dots within therectangle indicated by (A) are m/z and retention time of the mass peakof Marker A detected from the serum of the individual subject usingreverse phase chromatography. The dots in a cluster can be regarded asthe same retention time and the same m/z in the error range, and thedots in a cluster are defined to be derived from the same peptide.

FIG. 2 illustrates the results of differential analysis in the case ofMarker A. As shown in the amino acid sequences resulting of MS/MSanalysis in FIG. 4, Marker A is Complement C3-derived peptides CO3. FIG.2 shows the comparison between NDC and cognitive impairment (AD, MCI,DLB and FTD) related to CO3.

FIG. 3 illustrates the ROC curves of CO3 expressed by SEQ ID NO: 2.Definition of the ROC curve, see the section on the results of Example.FIG. 3A) shows the ROC curve of the comparison of AD vs. NDC. FIG. 3B)shows the ROC curve of the comparison of MCI vs. NDC.

FIG. 4 illustrates the MS/MS spectrum of CO3 by TOF/TOF massspectrometer. In FIG. 4 top, it was shown the amino acid sequence ofCO3, and it was shown y-ions and b-ions that appear in the MS/MSspectrum.

FIG. 5 illustrates the results of differential analysis of AP2Cexpressed by SEQ ID NO: 4. This figure shows a comparison betweencognitive impairment patients (AD, MCI, DLB, FTD) and subjects notsuffering from psychiatry disease (NDC).

FIG. 6 illustrates the results of differential analysis of SYN3expressed by SEQ ID NO: 6. This figure shows a comparison betweencognitive impairment patients (AD, MCI, DLB, FTD) and subjects notsuffering from psychiatry disease (NDC).

FIG. 7 illustrates the results of differential analysis of OXYRexpressed by SEQ ID NO: 8. This figure shows a comparison betweencognitive impairment patients (AD, MCI, DLB, FTD) and subjects notsuffering from psychiatry disease (NDC).

FIG. 8 illustrates the results of differential analysis of ITH5Lexpressed by SEQ ID NO: 10. This figure shows a comparison betweencognitive impairment patients (AD, MCI, DLB, FTD) and subjects notsuffering from psychiatry disease (NDC).

FIG. 9 illustrates the results of differential analysis of HERC2expressed by SEQ ID NO: 12. This figure shows a comparison betweencognitive impairment patients (AD, MCI, DLB, FTD) and subjects notsuffering from psychiatry disease (NDC).

FIG. 10 illustrates the results of differential analysis of THRBexpressed by SEQ ID NO: 14. This figure shows a comparison betweencognitive impairment patients (AD, MCI, DLB, FTD) and subjects notsuffering from psychiatry disease (NDC).

FIG. 11 illustrates the results of differential analysis of TTHYexpressed by SEQ ID NO: 16. This figure shows a comparison betweencognitive impairment patients (AD, MCI, DLB, FTD) and subjects notsuffering from psychiatry disease (NDC).

FIG. 12 illustrates the results of differential analysis of TNR16expressed by SEQ ID NO: 18. This figure shows a comparison betweencognitive impairment patients (AD, MCI, DLB, FTD) and subjects notsuffering from psychiatry disease (NDC).

FIG. 13 illustrates the results of differential analysis of C04-1expressed by SEQ ID NO: 20. This figure shows a comparison betweencognitive impairment patients (AD, MCI, DLB, FTD) and subjects notsuffering from psychiatry disease (NDC).

FIG. 14 illustrates the results of differential analysis of C04-2expressed by SEQ ID NO: 22. This figure shows a comparison betweencognitive impairment patients (AD, MCI, DLB, FTD) and subjects notsuffering from psychiatry disease (NDC).

FIG. 15 illustrates the results of differential analysis of FIBA-1expressed by SEQ ID NO: 24. This figure shows a comparison betweencognitive impairment patients (AD, MCI, DLB, FTD) and subjects notsuffering from psychiatry disease (NDC).

FIG. 16 illustrates the results of differential analysis of FIBA-2expressed by SEQ ID NO: 26. This figure shows a comparison betweencognitive impairment patients (AD, MCI, DLB, FTD) and subjects notsuffering from psychiatry disease (NDC).

FIG. 17 illustrates the results of differential analysis of FIBA-3expressed by SEQ ID NO: 27. This figure shows a comparison betweencognitive impairment patients (AD, MCI, DLB, FTD) and subjects notsuffering from psychiatry disease (NDC).

DESCRIPTION OF EMBODIMENTS

The present invention is a method for determining the kind and theamount of intact protein and/or its partial peptide when test subject issuffering from cognitive impairment as well as for diagnosing whethertest subject is suffering from cognitive impairment. A peptide isgenerally said to be a chemical entity, made by polymerizing a number ofamino acids, of less than 10,000 in molecular weight or by polymerizingseveral to less than about 50 amino acid residues. While in the presentinvention a partial peptide of an intact protein can be used as abiomarker for detection of cognitive impairment, such partial peptide isdefined as a peptide of less than 10,000 in molecular weight consistingof a part of the amino acid sequence of the intact protein. Such peptidemay arise as a partial peptide during the expression by transcriptionfollowed by synthesis by translation before maturing into an intactprotein or as a peptide produced by enzyme digestion in the body afterthe intact protein has been synthesized. It is possible that, when thebody is in abnormal state suffering from such disease as cognitiveimpairment, the mechanism for protein synthesis and regulation isde-regulated. In other words, the present invention is also a method fordetermining if test subject is in normal state or is suffering fromcognitive impairment by using the degree of protein synthesis and/orprotein digestion as an indicator. The detection of cognitive impairmentin the present invention means evaluation and differentiation, i.e.,diagnosis of test subject as to whether the subject is suffering fromcognitive impairment. The present invention can also include theevaluation of patient's risk of suffering from more serious cognitiveimpairment.

Specifically, in the method of the present invention, the examples ofintact protein that can be used as a cognitive impairment includeComplement C3 consisting of amino acid sequence expressed by SEQ ID NO:1, Transcription factor AP-2 gamma consisting of amino acid sequenceexpressed by SEQ ID NO: 3, Synapsin-3 consisting of amino acid sequenceexpressed by SEQ ID NO: 5, Oxytocin receptor consisting of amino acidsequence expressed by SEQ ID NO: 7, Inter-alpha-trypsin inhibitor heavychain H5-like protein consisting of amino acid sequence expressed by SEQID NO: 9, E3 ubiquitin-protein ligase HERC2 consisting of amino acidsequence expressed by SEQ ID NO: 11, Prothrombin consisting of aminoacid sequence expressed by SEQ ID NO: 13, Transthyretin consisting ofamino acid sequence expressed by SEQ ID NO: 15, Tumor necrosis factorreceptor superfamily member 16 consisting of amino acid sequenceexpressed by SEQ ID NO: 17, Complement C4-A consisting of amino acidsequence expressed by SEQ ID NO: 19, Complement C4-B consisting of aminoacid sequence expressed by SEQ ID NO: 21, Fibrinogen alpha chain(isoform 1) consisting of amino acid sequence expressed by SEQ ID NO:23, and Fibrinogen alpha chain (isoform 2) consisting of amino acidsequence expressed by SEQ ID NO: 25, and further, the peptide fragmentsthat comprise of partial peptides of not less than 5 amino acid residuesof these intact proteins can be used as same purpose.

Still further, an example of biomarkers for cognitive impairment of thepresent invention includes the partial peptides consisting of amino acidsequence expressed by SEQ ID NO: 2 of Complement C3-derived peptide CO3,SEQ ID NO: 4 of Transcription factor AP-2 gamma-derived peptide AP2C,SEQ ID NO: 6 of Synapsin-3-derived peptide SYN3, SEQ ID NO: 8 ofOxytocin receptor-derived peptide OXYR, SEQ ID NO: 10 ofInter-alpha-trypsin inhibitor heavy chain H5-like protein-derivedpeptide ITH5L, SEQ ID NO: 12 of E3 ubiquitin-protein ligaseHERC2-derived peptide HERC2, SEQ ID NO: 14 of Prothrombin-derivedpeptide THRB, SEQ ID NO: 16 of Transthyretin-derived peptide TTHY, SEQID NO: 18 of Tumor necrosis factor receptor superfamily member16-derived peptide TNR16, SEQ ID NO: 20 of Complement C4-derived peptideC04-1, SEQ ID NO: 22 of Complement C4-derived peptide C04-2, SEQ ID NO:24 of Fibrinogen alpha chain-derived peptide FIBA-1, SEQ ID NO: 26 ofFibrinogen alpha chain-derived peptide FIBA-2, and SEQ ID NO: 27 ofFibrinogen alpha chain-derived peptide FIBA-3. In the present invention,proteins and peptides consisting of amino acid sequences derived fromSEQ ID NOS: 1 through 27 by deletion, exchange, and/or addition of oneor a few amino acids can be used as biomarkers and are included in thepresent invention. ‘”One or a few” herein means “one or three,” “one ortwo,” or “one.” Furthermore, the partial peptides that can be used asbiomarkers in the present invention include those peptide fragmentsconsisting of not less than 5 amino acid residues arising respectivelyfrom SEQ ID NOS: 1 through 27. The basis for the limitation of peptidefragments consisting of not less than 5 amino acid residues is in thedescription below in Non-patent Document 2. The document reported thatan antibody obtained by using the peptide IRGERA as immunogen, which wasthe C-terminus (130-135) of histone H3, recognized the peptide IKGERAderived by exchange of K for R and the peptide CGGGERA which was derivedby deletion of IR followed by addition of CGG. This demonstrates thatthe immunogenicity (antigenicity) is recognized by a peptide of not lessthan 4 amino acid residues. In order to expand this finding to otherpeptides than the C-terminus of histone H3, the number of amino acidresidue is defined as not less than 5 instead of 4 in the presentinvention. To make such a low molecular weight peptide as the subject ofthe present invention is important when the method of detection anddifferentiation uses immunological means including immunoblot, ELISA andimmunoMS.

It is to be noted that there are cases where a sugar chain or sugarchains have been added to an intact protein or its partial peptide toform glycated entities. Proteins and partial peptides in glycated formcan also be used as biomarkers for detection of cognitive impairment.

It is also to be noted that, in the present invention, biomarker can bequantified or its presence or absence can be determined qualitatively.

Two-dimensional electrophoresis (2-DE) or 2-dimensional chromatography(2-DC) can be used in the present invention to separate biomarkers inbiological materials including serum. Known chromatographic methods canbe selected from ion-exchange chromatography, reverse-phasechromatography and gel-filtration chromatography. It is also possible tomake quantification with the SRM/MRM method in LC-MS/MS technology.Furthermore, the immunoMS method which these inventors have developed,where target protein or peptide is captured by beads (including magneticones) with antibody linked to the protein or peptide, eluted from thebeads, and determined by mass spectrometry enables convenientdetermination of presence or absence or the amount of target protein,protein fragment or peptide without the use of 2-DE or chromatography.

It is possible with the use of the method disclosed in the presentinvention to evaluate at the stage of mild of cognitive dysfunction intest subject and therefore it can be useful in prophylactic medicine.Further, when psychotherapy and/or drug therapy is given to patientswith cognitive impairment, it is reflected in the amount of proteins andpartial peptides in biological materials such as serum if theprogression of the disorder has been inhibited. Therefore, by measuringthese proteins and partial peptides, it is possible to evaluate anddetermine therapeutic effect.

The kind and amount of a protein in biological materials can bedetermined by various methods. If target protein (including proteinfragment and partial peptide) has been characterized and when anantibody (primary antibody) to it has already been obtained, thefollowing methods can be used:

1. Immunoblot

This is one of the simplest methods. Test serum in a fixed amount (about1 microliter) after stepwise dilution is dropped onto an appropriatemembrane such as of nitrocellulose and dried in air. The membrane istreated with a blocking solution containing a protein such as BSA,washed, reacted with primary antibody, and washed. Thereafter, themembrane is reacted with labeled secondary antibody to detect theprimary antibody. The membrane is washed and the label is visualized tomeasure its density.

2. Western Blotting

After separation with one-dimensional or two-dimensional electrophoresisinvolving isoelectric focusing or SDS-PAGE, proteins are transferredonto such an appropriate membrane as of PVDF and their amounts aredetermined, as in above-mentioned immunoblot, using primary antibody andlabeled secondary antibody.

3. ELISA

Antibody to protein or its partial peptide is fixed to such a plate as achemically modified microtiter plate. Appropriate amounts of samplesafter stepwise dilution are applied to the plate and incubated. Proteinsand peptides not captured are removed by washing. Next, the plate isincubated with secondary antibody labeled with fluorescent orchemiluminescent substance or enzyme. After addition of respectivesubstrate, fluorescence, chemiluminescence or visible light due toenzyme reaction is measured for evaluation and judgment.

Additional examples of methods are illustrated below (see PatentDocument 2) but the invention is not limited by these examples.

4. Methods that Use Microarray (Microchip)

A microarray is a general term for devices where solidified materialswith affinity for target substances are arrayed on solid support(plate). In the present invention, antibodies or aptamer to proteins andpartial peptides are arrayed. A sample of biological material is placedon the microarray for fixation of target proteins or partial peptidesand the microarray is then incubated with secondary antibody labeledwith fluorescent or chemiluminescent substance or enzyme. After additionof respective substrate, fluorescence, chemiluminescence or visiblelight due to enzyme reaction is measured.

5. Mass Spectrometry

In mass spectrometry, for example, antibody to a specified protein orpartial peptide is attached to chemically modified microbeads or plate(protein chip). The microbeads could be magnetic beads. There are norequirements for the material of the plate. The antibody to be usedcould be (1) an antibody which recognizes the full length form of thespecified protein only, (2) an antibody which recognizes a partialpeptide only, (3) all of antibodies which recognizes both the specifiedprotein and its partial peptide, or a combination of (1) and (2), (1)and (3), or (2) and (3). Samples after stepwise dilution with originalsolvent or buffer are added to the microbeads or plate carrying antibodyor antibodies and incubated. Those proteins and partial peptides notcaptured are removed by washing. The protein or partial peptide capturedby microbeads or plate is eluted, and analyzed by mass spectrometry withMALDI-TOF-MS, SELDI-TOF-MS, etc. Measurements are made with respect tothe mass and intensity of the peak due to the protein, protein fragmentor partial peptide. Prior to the measurements a fixed amount ofsubstance serving as the internal standard is added to the originalbiological material and the intensity of its peak is also measured. Theconcentration of the target in the original biological material can becalculated from the ratio of peak intensity of the target to the peakintensity of the internal standard. This is called immunoMS method.Further, it is possible to make quantification, after the sample isdiluted with original solvent or buffer, or after part of proteins areremoved, by separation with HPLC followed by mass spectrometry withelectrospray ionization (ESI) method. Therein the SRM/MRM method can beutilized for absolute quantification with the use of an isotope-labeledinternal standard peptide.

Furthermore, in addition to the above-mentioned methods, it is possibleto analyze proteins and partial peptides by using 2-DE, surface plasmonresonance, etc.

The present invention includes the method to detect cognitive impairmentfrom the presence or absence or amount of the above-mentioned biomarkerafter applying biological material obtained from test subject to 2-DE orsurface plasmon resonance.

EXAMPLES

Discovery of a marker peptide for detection of cognitive impairmentusing two-dimensional liquid chromatography-mass spectrometry(2D-LC-MALDI TOF-MS).

(1) Serum Samples.

Followings, the characters before the parenthesis are an abbreviation.

A sera obtained from 40 AD (Alzheimer's disease), 35MCI (mild cognitiveimpairment), 13 DLB (Dementia with Lewy bodies), 7 FTD (frontotemporallobar degeneration), and 21 NDC (subjects not suffering from psychiatrydisease) were used.

(2) Methods

After 475 μl of 0.1% trifluoroacetic acid (TFA) were added in each of 25μl of sera, samples were boiled for 15 min at 100 degrees. Subsequently,in order to recover peptides of molecular weight of 10,000 or less,ultrafiltration were performed by using YM-10 filter unit (MilliporeCorp.). Then the analysis using 2D-LC-MALDI TOF-MS were performed asfollows. In other words, recovering samples were fractionated to 382fractions per sample by using two-dimensional HPLC (SCX cation exchangecolumn at one-dimension and C18 reverse-phase column at two-dimension).The samples were fractionated into two fractions by SCX cation exchangecolumn, namely, SCX 1 fraction is through fraction, SCX 2 fraction isthe fraction that eluted with 100% salt solution. Two fractions thatwere fractionated by SCX, respectively, were fractionated 191 fractionsby C18 reverse phase column chromatography. It was eluted with 6 secondsin one fraction, and the retention times were calculated by multiplyingthe number of minus 1 from number of eluted fractions to 6 seconds. Allfractionated samples were spotted on MALDI target plate (MTP AnchorChip™600/384 plate, BRUKER DALTONICS) for MALDI TOF/TOF mass spectrometer(ultraflex TOF/TOF, BRUKER DALTONICS) using a spotting robot (AccuSpot,SHIMADZU) that is connected online, and matrix solution(alpha-cyano-hydroxycinnamic acid, CHCA) were mixed and crystallized.After mounting MALDI target plate into ultraflex TOF/TOF, the mass andthe peak area of the mass were measured automatically in reflectron modeby irradiating to crystallized sample by laser. Peak area was normalizedwith 250 mole of per each well of bradykinin 1-7 fragment that was addedinto matrix solution in advance. In other words, the area value wascalculated dividing the peak area of specific mass in sample by the peakarea obtained from bradykinin1-7 fragment. This area value iscorresponding in 25 μl of sample serum. Detection of difference inabundance of peptides in serum between groups (called differentialanalysis) was performed using multi-group statistical analysis softwareParnassum™ (MCBI) developed by us. Peptide that was observed todifference in abundance was directly determined amino acid sequence inMS/MS analysis by ultraflex TOF/TOF, and intact proteins or peptides oftheir origin were identified.

(3) Results

The following shows the result of differential analysis by Parnassumsoftware for data of serum individual subjects obtained using 2D-LCMALDI TOF-MS. FIG. 1 shows the result that was obtained from sample thatwas applied to 2D-LC-MALDI TOF-MS. Sample was fractionated into 2fractions by SCX cation exchange column in the first dimension, thenfirst fractions from SCX column (SCX 1) were fractionated into 191fractions by C18 reverse-phase column. Mass spectra of 191 fractionswere obtained by MALDI TOF-MS measuring. As the horizontal axis is them/z and the vertical axis is the fractions of reverse-phase columnchromatography, FIG. 1 was visualized by Parnassum software developed bypresent inventors. The dots in FIG. 1 shows respectively TOF-MS peakderived from the individual subject. The sections that dots are gatheredcan be regarded as the same retention time and the same m/z in the errorrange, and the dots in the sections are defined to be derived from thesame peptide. These sections are referred to as clusters. Section (A) ofFIG. 1 shows cluster of Marker A.

FIG. 2 shows the results of differential analysis in the case of MarkerA. As shown in FIG. 4, Marker A is Complement C3-derived peptides CO3.FIG. 2 shows the comparison between subjects not suffering frompsychiatry disease (NDC) and cognitive impairment (AD, MCI, DLB and FTD)related to CO3. In the results of t-test, area values of cognitiveimpairment (AD, MCI, DLB and FTD) were significantly higher than NDC(p<0.05).

From the results of FIG. 2, in order to evaluate the extent to which theMarker A is useful as biomarker, the analysis by receiver operatingcharacteristic (ROC) curve was performed. A) and B) in FIG. 3 showsrespectively the ROC curve of the comparison of AD vs. NDC and MCI vs.NDC. If the area value (hereinafter referred to as the AUC value) ofunder the ROC curve is close to 1, the usefulness as biomarker of MarkerA will be higher. In A) and B) of FIG. 3, the typical values ofsensitivity and specificity are the values of the point (open square inthe figure) of the coordinate on ROC curve that the distance isminimized when a straight line was drawn to ROC curve from the point of100% on y-axis. The value of cut-off giving this point becomes a usefulthreshold to distinguish between the different groups, and the values ofsensitivity and specificity at that time (i.e., above the typicalvalues) becomes an indicator of the usefulness of biomarkers togetherwith AUC values. In A) of FIG. 3, as typical values in AD vs. NDC, thesensitivity was 73.0%, the specificity was 100%, and the AUC value was0.88. In B) of FIG. 3, as typical values in MCI vs. NDC, the sensitivitywas 70.6%, the specificity was 89.5%, and the AUC value was 0.83.

Thus, it was revealed that Marker A was useful to distinguish AD and MCIwith NDC. In particular, since MCI is the state of previous stage of AD,Marker A is considered to be an extremely useful marker to detect MCIfor early diagnosis of potential subjects to migrate to AD.

FIG. 4, for Marker A, illustrates the results of MS/MS spectrum usingultraflex TOF/TOF. The signals that show y-ions and b-ions have enoughappeared, and the amino acid sequence could be readily identified.Mascot search was performed on this result and the protein of origin orthe peptide (hereinafter referred to as intact proteins or peptides) isComplement C3, and the detected peptide was found that the sequence isAPVIHQEMIGGLRN (SEQ ID NO: 2). CO3 of entry name of Swiss-Prot againstComplement C3 will use as an abbreviation of the peptide name.Followings, for peptides other than CO3, entry name will use as peptidename, similarly.

Including the Marker A, the peptides that have difference in abundancebetween the groups in serum were measured MS/MS spectra using ultraflexTOF/TOF, and in addition to determining the amino acid sequence, theresults identified intact proteins or peptides were shown below. Forpeptides other than Marker A, the signals that show y-ions and b-ionshas enough appeared, and the amino acid sequence could be readilyidentified. The following amino acid sequence that shows a set of twosequences, the first sequence shows the amino acid sequence of intactproteins, and the second sequence shows the amino acid sequence ofpeptide detected by 2D-LC MALDI TOF-MS. The peptide comprising of theunderlined portion in the first sequence correspond to the sequence ofpeptide detected by 2D-LC MALDI TOF-MS. The amino acid sequence startingat 0001 in the sequence shows the sequence of the N-terminus side.

(1) Complement C3-Derived Peptide CO3

CO3 shown as SEQ ID NO: 2 had formed a cluster by clustering usingParnassum software.

As shown in FIG. 2, area values of cognitive impairment (AD, MCI, DLBand FTD) were significantly higher than NDC (t-test, p<0.05). Thus, itwas revealed that CO3 shown as SEQ ID NO: 2 was useful to distinguishpatient of cognitive impairment (AD, MCI, DLB and FTD) with subjects notsuffering from psychiatry disease (NDC). According to the analysis byreceiver operating characteristic (ROC) curve, CO3 was clearly useful todistinguish AD and MCI with NDC. (See FIG. 3A), 3B) and Table 1).

Intact Protein/Peptide

(SEQ ID NO: 1) 0001 SPMYSIITPN ILRLESEETM VLEAHDAQGD VPVTVTVHDFPGKKLVLSSE 0051 KTVLTPATNH MGNVTFTIPA NREFKSEKGR NKFVTVQATF GTQVVEKVVL0101 VSLQSGYLFI QTDKTIYTPG STVLYRIFTV NHKLLPVGRT VMVNIENPEG 0151IPVKQDSLSS QNQLGVLPLS WDIPELVNMG QWKIRAYYEN SPQQVFSTEF 0201 EVKEYVLPSFEVIVEPTEKF YYIYNEKGLE VTITARFLYG KKVEGTAFVI 0251 FGIQDGEQRI SLPESLKRIPIEDGSGEVVL SRKVLLDGVQ NPRAEDLVGK 0301 SLYVSATVIL HSGSDMVQAE RSGIPIVTSPYQIHFTKTPK YFKPGMPFDL 0351 MVFVTNPDGS PAYRVPVAVQ GEDTVQSLTQ GDGVAKLSINTHPSQKPLSI 0401 TVRTKKQELS EAEQATRTMQ ALPYSTVGNS NNYLHLSVLR TELRPGETLN0451 VNFLLRMDRA HEAKIRYYTY LIMNKGRLLK AGRQVREPGQ DLVVLPLSIT 0501TDFIPSFRLV AYYTLIGASG QREVVADSVW VDVKDSCVGS LVVKSGQSED 0551 RQPVPGQQMTLKIEGDHGAR VVLVAVDKGV FVLNKKNKLT QSKIWDVVEK 0601 ADIGCTPGSG KDYAGVFSDAGLTFTSSSGQ QTAQRAELQC PQPAARRRRS 0651 VQLTEKRMDK VGKYPKELRK CCEDGMRENPMRFSCQRRTR FISLGEACKK 0701 VFLDCCNYIT ELRRQHARAS HLGLARSNLD EDIIAEENIVSRSEFPESWL 0751 WNVEDLKEPP KNGISTKLMN IFLKDSITTW EILAVSMSDK KGICVADPFE0801 VTVMQDFFID LRLPYSVVRN EQVEIRAVLY NYRQNQELKV RVELLHNPAF 0851CSLATTKRRH QQTVTIPPKS SLSVPYVIVP LKTGLQEVEV KAAVYHHFIS 0901 DGVRKSLKVVPEGIRMNKTV AVRTLDPERL GREGVQKEDI PPADLSDQVP 0951 DTESETRILL QGTPVAQMTEDAVDAERLKH LIVTPSGCGE QNMIGMTPTV 1001 IAVHYLDETE QWEKFGLEKR QGALELIKKGYTQQLAFRQP SSAFAAFVKR 1051 APSTWLTAYV VKVFSLAVNL IAIDSQVLCG AVKWLILEKQKPDGVFQEDA 1101 PVIHQEMIGG LRNNNEKDMA LTAFVLISLQ EAKDICEEQV NSLPGSITKA1151 GDFLEANYMN LQRSYTVAIA GYALAQMGRL KGPLLNKFLT TAKDKNRWED 1201PGKQLYNVEA TSYALLALLQ LKDFDFVPPV VRWLNEQRYY GGGYGSTQAT 1251 FMVFQALAQYQKDAPDHQEL NLDVSLQLPS RSSKITHRIH WESASLLRSE 1301 ETKENEGFTV TAEGKGQGTLSVVTMYHAKA KDQLTCNKFD LKVTIKPAPE 1351 TEKRPQDAKN TMILEICTRY RGDQDATMSILDISMMTGFA PDTDDLKQLA 1401 NGVDRYISKY ELDKAFSDRN TLIIYLDKVS HSEDDCLAFKVHQYFNVELI 1451 QPGAVKVYAY YNLEESCTRF YHPEKEDGKL NKLCRDELCR CAEENCFIQK1501 SDDKVTLEER LDKACEPGVD YVYKTRLVKV QLSNDFDEYI MAIEQTIKSG 1551SDEVQVGQQR TFISPIKCRE ALKLEEKKHY LMWGLSSDFW GEKPNLSYII 1601 GKDTWVEHWPEEDECQDEEN QKQCQDLGAF TESMVVFGCP N

Complement C3-Derived Peptide CO3

(SEQ ID NO: 2) APVIHQEMIGGLRN

(2) Transcription Factor AP-2 Gamma-Derived Peptide AP2C

For AP2C shown as SEQ ID NO: 4, area values of cognitive impairment (AD,MCI, DLB and FTD) were significantly higher than NDC. (t-test, p<0.05)(see FIG. 5)

Thus, it was revealed that AP2C shown as SEQ ID NO: 4 was useful todistinguish patient of cognitive impairment (AD, MCI, DLB and FTD) withsubjects not suffering from psychiatry disease (NDC). According to theanalysis by receiver operating characteristic (ROC) curve, CO3 wasclearly useful to distinguish AD and MCI with NDC. (See Table 1)

Intact Protein/Peptide

(SEQ ID NO: 3) 0001 MLWKITDNVK YEEDCEDRHD GSSNGNPRVP HLSSAGQHLYSPAPPLSHTG 0051 VAEYQPPPYF PPPYQQLAYS QSADPYSHLG EAYAAAINPL HQPAPTGSQQ0101 QAWPGRQSQE GAGLPSHHGR PAGLLPHLSG LEAGAVSARR DAYRRSDLLL 0151PHAHALDAAG LAENLGLHDM PHQMDEVQNV DDQHLLLHDQ TVIRKGPISM 0201 TKNPLNLPCQKELVGAVMNP TEVFCSVPGR LSLLSSTSKY KVTVAEVQRR 0251 LSPPECLNAS LLGGVLRRAKSKNGGRSLRE KLDKIGLNLP AGRRKAAHVT 0301 LLTSLVEGEA VHLARDFAYV CEAEFPSKPVAEYLTRPHLG GRNEMAARKN 0351 MLLAAQQLCK EFTELLSQDR TPHGTSRLAP VLETNIQNCLSHFSLITHGF 0401 GSQAICAAVS ALQNYIKEAL IVIDKSYMNP GDQSPADSNK TLEKMEKHRK

Transcription Factor AP-2 Gamma-Derived Peptide AP2C

(SEQ ID NO: 4) PGRQSQEGAGLPSHHG

(3) Synapsin-3-Derived Peptide SYN3

For SYN3 shown as SEQ ID NO: 6, area values of cognitive impairment (AD,MCI, DLB and FTD) were significantly higher than NDC. (t-test, p<0.05)(see FIG. 6)

Thus, it was revealed that SYN3 shown as SEQ ID NO: 6 was useful todistinguish patient of cognitive impairment (AD, MCI, DLB and FTD) withsubjects not suffering from psychiatry disease (NDC). According to theanalysis by receiver operating characteristic (ROC) curve, CO3 wasclearly useful to distinguish AD and MCI with NDC. (See Table 1)

Intact Protein/Peptide

(SEQ ID NO: 5) 0001 MNFLRRRLSD SSFMANLPNG YMTDLQRPDS STSSPASPAMERRHPQPLAA 0051 SFSSPGSSLF SSLSSAMKQA PQATSGLMEP PGPSTPIVQR PRILLVIDDA0101 HTDWSKYFHG KKVNGEIEIR VEQAEFSELN LAAYVTGGCM VDMQVVRNGT 0151KVVSRSFKPD FILVRQHAYS MALGEDYRSL VIGLQYGGLP AVNSLYSVYN 0201 FCSKPWVFSQLIKIFHSLGP EKFPLVEQTF FPNHKPMVTA PHFPVVVKLG 0251 HAHAGMGKIK VENQLDFQDITSVVAMAKTY ATTEAFIDSK YDIRIQKIGS 0301 NYKAYMRTSI SGNWKANTGS AMLEQVAMTERYRLWVDSCS EMFGGLDICA 0351 VKAVHSKDGR DYIIEVMDSS MPLIGEHVEE DRQLMADLVVSKMSQLPMPG 0401 GTAPSPLRPW APQIKSAKSP GQAQLGPQLG QPQPRPPPQG GPRQAQSPQP0451 QRSGSPSQQR LSPQGQQPLS PQSGSPQQQR SPGSPQLSRA SSGSSPNQAS 0501KPGATLASQP RPPVQGRSTS QQGEESKKPA PPHPHLNKSQ SLTNSLSTSD 0551 TSQRGTPSEDEAKAETIRNL RKSFASLFSD

Synapsin-3-Derived Peptide SYN3

(SEQ ID NO: 6) EMFGGLDICAVKAVHSK

(4) Oxytocin Receptor-Derived Peptide OXYR

For OXYR shown as SEQ ID NO: 8, area values of cognitive impairment (AD,MCI, DLB and FTD) were significantly higher than NDC. (t-test, p<0.05)(see FIG. 7) Thus, it was revealed that OXYR shown as SEQ ID NO: 8 wasuseful to distinguish patient of cognitive impairment (AD, MCI, DLB andFTD) with subjects not suffering from psychiatry disease (NDC).According to the analysis by receiver operating characteristic (ROC)curve, CO3 was clearly useful to distinguish AD and MCI with NDC. (SeeTable 1)

Intact Protein/Peptide

(SEQ ID NO: 7) 0001 MEGALAANWS AEAANASAAP PGAEGNRTAG PPRRNEALARVEVAVLCLIL 0051 LLALSGNACV LLALRTTRQK HSRLFFFMKH LSIADLVVAV FQVLPQLLWD0101 ITFRFYGPDL LCRLVKYLQV VGMFASTYLL LLMSLDRCLA ICQPLRSLRR 0151RTDRLAVLAT WLGCLVASAP QVHIFSLREV ADGVFDCWAV FIQPWGPKAY 0201 ITWITLAVYIVPVIVLAACY GLISFKIWQN LRLKTAAAAA AEAPEGAAAG 0251 DGGRVALARV SSVKLISKAKIRTVKMTFII VLAFIVCWTP FFFVQMWSVW 0301 DANAPKEASA FIIVMLLASL NSCCNPWIYMLFTGHLFHEL VQRFLCCSAS 0351 YLKGRRLGET SASKKSNSSS FVLSHRSSSQ RSCSQPSTA

Oxytocin Receptor-Derived Peptide OXYR

(SEQ ID NO: 8) AAPPGAEGNRT

(5) Inter-Alpha-Trypsin Inhibitor Heavy Chain H5-like Protein-DerivedPeptide ITH5L

For ITH5L shown as SEQ ID NO: 10, area values of cognitive impairment(AD, MCI and DLB) were significantly higher than NDC. (t-test, p<0.05)(see FIG. 8)

Thus, it was revealed that ITH5L shown as SEQ ID NO: 10 was useful todistinguish patient of cognitive impairment (AD, MCI and DLB) withsubjects not suffering from psychiatry disease (NDC). According to theanalysis by receiver operating characteristic (ROC) curve, CO3 wasclearly useful to distinguish AD and MCI with NDC. (See Table 1)

Intact Protein/Peptide

(SEQ ID NO: 9) 0001 GPPVPASSST KLLMTSYSMR STVVSRYAHT LVTSVLFNPHAEAHEAIFDL 0051 DLPHLAFISN FTMTINNKVY IAEVKEKHQA KKIYEEAHQQ GKTAAHVGIR0101 DRESEKFRIS TSLAAGTEVT FSLAYEELLQ RHQGQYQLVV SLRPGQLVKR 0151LSIEVTVSER TGISYVHIPP LRTGRLRTNA HASEVDSPPS TRIERGETCV 0201 RITYCPTLQDQSSISGSGIM ADFLVQYDVV MEDIIGDVQI YDDYFIHYFA 0251 PRGLPPMEKN VVFVIDVSSSMFGTKMEQTK TAMNVILSDL QANDYFNIIS 0301 FSDTVNVWKA GGSIQATIQN VHSAKDYLHCMEADGWTDVN SALLAAASVL 0351 NHSNQEPGRG PSVGRIPLII FLTDGEPTAG VTTPSVILSNVRQALGHRVS 0401 LFSLAFGDDA DFTLLRRLSL ENRGIARRIY EDTDAALQLK GLYEEISMPL0451 LADVRLNYLG GLVGASPWAV FPNYFGGSEL VVAGQVQPGK QELGIHLAAR 0501GPKDQLLVAH HSEGATNNSQ KAFGCPGEPA PNVAHFIRRL WAYVTIGELL 0551 DAHFQARDTTTRHLLAAKVL NLSLEYNFVT PLTSLVMVQP KQASEETRRQ 0601 TSTSAGPDTI MPSSSSRHGLGVSTAQPALV PKVISPKSRP VKPKFYLSST 0651 TTASTKKMLS SKELEPLGES PHTLSMPTYPKAKIPAQQDS GTLAQPTLRT 0701 KPTILVPSNS GTLLPLKPGS LSHQNPDILP TNSRTQVPPVKPGIPASPKA 0751 DTVKCVTPLH SKPGAPSHPQ LGALTSQAPK GLPQSRPGVS TLQVPKYPLH0801 TRPRVPAPKT RNNMPHLGPG ILLSKTPKIL LSLKPSAPPH QISTSISLSK 0851PETPNPHMPQ TPLPPRPDRP RPPLPESLST FPNTISSSTG PSSTTTTSVL 0901 GEPLPMPFTPTLPPGRFWHQ YDLLPGPQRT RQVLGPSRPG VPTMSLLNSS 0951 RPTPEGSPPN LPILLPSSILPEAISLLLLP EELELLSESM VESKFVESLN 1001 PPAFYTFLTP DEDGSPNWDG NSEEILGGAGGSMESQGSSV GLAKGTLPSI 1051 FTFSSSVDGD PHFVIQIPHS EEKICFTLNG HPGDLLQLIEDPKAGLHVSG 1101 KLLGAPPRPG HKDQTRTYFQ IITVTTDKPR AYTITISRSS ISLRGEGTLR1151 LSWDQPALLK RPQLELYVAA AARLTLRLGP YLEFLVLRHR YRHPSTLQLP 1201HLGFYVANGS GLSPSARGLI GQFQHADIRL VTGPMGPCLR RHHGPDVPVI 1251 LGKRLLKDSPRLLPRWASCW LVKRSHVELL LGHPYLSYVLInter-Alpha-Trypsin Inhibitor Heavy Chain H5-like Protein-DerivedPeptide ITH5L

(SEQ ID NO: 10) RVSLFSLAFGDDAD

(6) E3 Ubiquitin-Protein Ligase HERC2-Derived Peptide HERC2

For HERC2 shown as SEQ ID NO: 12, area values of cognitive impairment(AD, MCI and DLB) were significantly higher than NDC. (t-test, p<0.05)(see FIG. 9)

Thus, it was revealed that HERC2 shown as SEQ ID NO: 12 was useful todistinguish patient of cognitive impairment (AD, MCI and DLB) withsubjects not suffering from psychiatry disease (NDC). According to theanalysis by receiver operating characteristic (ROC) curve, CO3 wasclearly useful to distinguish AD and MCI with NDC. (See Table 1)

Intact Protein/Peptide

(SEQ ID NO: 11) 0001 MPSESFCLAA QARLDSKWLK TDIQLAFTRD GLCGLWNEMVKDGEIVYTGT 0051 ESTQNGELPP RKDDSVEPSG TKKEDLNDKE KKDEEETPAP IYRAKSILDS0101 WVWGKQPDVN ELKECLSVLV KEQQALAVQS ATTTLSALRL KQRLVILERY 0151FIALNRTVFQ ENVKVKWKSS GISLPPVDKK SSRPAGKGVE GLARVGSRAA 0201 LSFAFAFLRRAWRSGEDADL CSELLQESLD ALRALPEASL FDESTVSSVW 0251 LEVVERATRF LRSVVTGDVHGTPATKGPGS IPLQDQHLAL AILLELAVQR 0301 GTLSQMLSAI LLLLQLWDSG AQETDNERSAQGTSAPLLPL LQRFQSIICR 0351 KDAPHSEGDM HLLSGPLSPN ESFLRYLTLP QDNELAIDLRQTAVVVMAHL 0401 DRLATPCMPP LCSSPTSHKG SLQEVIGWGL IGWKYYANVI GPIQCEGLAN0451 LGVTQIACAE KRFLILSRNG RVYTQAYNSD TLAPQLVQGL ASRNIVKIAA 0501HSDGHHYLAL AATGEVYSWG CGDGGRLGHG DTVPLEEPKV ISAFSGKQAG 0551 KHVVHIACGSTYSAAITAEG ELYTWGRGNY GRLGHGSSED EAIPMLVAGL 0601 KGLKVIDVAC GSGDAQTLAVTENGQVWSWG DGDYGKLGRG GSDGCKTPKL 0651 IEKLQDLDVV KVRCGSQFSI ALTKDGQVYSWGKGDNQRLG HGTEEHVRYP 0701 KLLEGLQGKK VIDVAAGSTH CLALTEDSEV HSWGSNDQCQHFDTLRVTKP 0751 EPAALPGLDT KHIVGIACGP AQSFAWSSCS EWSIGLRVPF VVDICSMTFE0801 QLDLLLRQVS EGMDGSADWP PPQEKECVAV ATLNLLRLQL HAAISHQVDP 0851EFLGLGLGSI LLNSLKQTVV TLASSAGVLS TVQSAAQAVL QSGWSVLLPT 0901 AEERARALSALLPCAVSGNE VNISPGRRFM IDLLVGSLMA DGGLESALHA 0951 AITAEIQDIE AKKEAQKEKEIDEQEANAST FHRSRTPLDK DLINTGICES 1001 SGKQCLPLVQ LIQQLLRNIA SQTVARLKDVARRISSCLDF EQHSRERSAS 1051 LDLLLRFQRL LISKLYPGES IGQTSDISSP ELMGVGSLLKKYTALLCTHI 1101 GDILPVAASI ASTSWRHFAE VAYIVEGDFT GVLLPELVVS IVLLLSKNAG1151 LMQEAGAVPL LGGLLEHLDR FNHLAPGKER DDHEELAWPG IMESFFTGQN 1201CRNNEEVTLI RKADLENHNK DGGFWTVIDG KVYDIKDFQT QSLTGNSILA 1251 QFAGEDPVVALEAALQFEDT RESMHAFCVG QYLEPDQEIV TIPDLGSLSS 1301 PLIDTERNLG LLLGLHASYLAMSTPLSPVE IECAKWLQSS IFSGGLQTSQ 1351 IHYSYNEEKD EDHCSSPGGT PASKSRLCSHRRALGDHSQA FLQAIADNNI 1401 QDHNVKDFLC QIERYCRQCH LTTPIMFPPE HPVEEVGRLLLCCLLKHEDL 1451 GHVALSLVHA GALGIEQVKH RTLPKSVVDV CRVVYQAKCS LIKTHQEQGR1501 SYKEVCAPVI ERLRFLFNEL RPAVCNDLSI MSKFKLLSSL PRWRRIAQKI 1551IRERRKKRVP KKPESTDDEE KIGNEESDLE EACILPHSPI NVDKRPIAIK 1601 SPKDKWQPLLSTVTGVHKYK WLKQNVQGLY PQSPLLSTIA EFALKEEPVD 1651 VEKMRKCLLK QLERAEVRLEGIDTILKLAS KNFLLPSVQY AMFCGWQRLI 1701 PEGIDIGEPL TDCLKDVDLI PPFNRMLLEVTFGKLYAWAV QNIRNVLMDA 1751 SAKFKELGIQ PVPLQTITNE NPSGPSLGTI PQARFLLVMLSMLTLQHGAN 1801 NLDLLLNSGM LALTQTALRL IGPSCDNVEE DMNASAQGAS ATVLEETRKE1851 TAPVQLPVSG PELAAMMKIG TRVMRGVDWK WGDQDGPPPG LGRVIGELGE 1901DGWIRVQWDT GSTNSYRMGK EGKYDLKLAE LPAAAQPSAE DSDTEDDSEA 1951 EQTERNIHPTAMMFTSTINL LQTLCLSAGV HAEIMQSEAT KTLCGLLRML 2001 VESGTTDKTS SPNRLVYREQHRSWCTLGFV RSIALTPQVC GALSSPQWIT 2051 LLMKVVEGHA PFTATSLQRQ ILAVHLLQAVLPSWDKTERA RDMKCLVEKL 2101 FDFLGSLLTT CSSDVPLLRE STLRRRRVRP QASLTATHSSTLAEEVVALL 2151 RTLHSLTQWN GLINKYINSQ LRSITHSFVG RPSEGAQLED YFPDSENPEV2201 GGLMAVLAVI GGIDGRLRLG GQVMHDEFGE GTVTRITPKG KITVQFSDMR 2251TCRVCPLNQL KPLPAVAFNV NNLPFTEPML SVWAQLVNLA GSKLEKHKIK 2301 KSTKQAFAGQVDLDLLRCQQ LKLYILKAGR ALLSHQDKLR QILSQPAVQE 2351 TGTVHTDDGA VVSPDLGDMSPEGPQPPMIL LQQLLASATQ PSPVKAIFDK 2401 QELEAAALAV CQCLAVESTH PSSPGFEDCSSSEATTPVAV QHIRPARVKR 2451 RKQSPVPALP IVVQLMEMGF SRRNIEFALK SLTGASGNASSLPGVEALVG 2501 WLLDHSDIQV TELSDADTVS DEYSDEEVVE DVDDAAYSMS TGAVVTESQT2551 YKKRADFLSN DDYAVYVREN IQVGMMVRCC RAYEEVCEGD VGKVIKLDRD 2601GLHDLNVQCD WQQKGGTYWV RYIHVELIGY PPPSSSSHIK IGDKVRVKAS 2651 VTTPKYKWGSVTHQSVGVVK AFSANGKDII VDFPQQSHWT GLLSEMELVP 2701 SIHPGVTCDG CQMFPINGSRFKCRNCDDFD FCETCFKTKK HNTRHTFGRI 2751 NEPGQSAVFC GRSGKQLKRC HSSQPGMLLDSWSRMVKSLN VSSSVNQASR 2801 LIDGSEPCWQ SSGSQGKHWI RLEIFPDVLV HRLKMIVDPADSSYMPSLVV 2851 VSGGNSLNNL IELKTININP SDTTVPLLND CTEYHRYIEI AIKQCRSSGI2901 DCKIHGLILL GRIRAEEEDL AAVPFLASDN EEEEDEKGNS GSLIRKKAAG 2951LESAATIRTK VFVWGLNDKD QLGGLKGSKI KVPSFSETLS ALNVVQVAGG 3001 SKSLFAVTVEGKVYACGEAT NGRLGLGISS GTVPIPRQIT ALSSYVVKKV 3051 AVHSGGRHAT ALTVDGKVFSWGEGDDGKLG HFSRMNCDKP RLIEALKTKR 3101 IRDIACGSSH SAALTSSGEL YTWGLGEYGRLGHGDNTTQL KPKMVKVLLG 3151 HRVIQVACGS RDAQTLALTD EGLVFSWGDG DFGKLGRGGSEGCNIPQNIE 3201 RLNGQGVCQI ECGAQFSLAL TKSGVVWTWG KGDYFRLGHG SDVHVRKPQV3251 VEGLRGKKIV HVAVGALHCL AVTDSGQVYA WGDNDHGQQG NGTTTVNRKP 3301TLVQGLEGQK ITRVACGSSH SVAWTTVDVA TPSVHEPVLF QTARDPLGAS 3351 YLGVPSDADSSAASNKISGA SNSKPNRPSL AKILLSLDGN LAKQQALSHI 3401 LTALQIMYAR DAVVGALMPAAMIAPVECPS FSSAAPSDAS AMASPMNGEE 3451 CMLAVDIEDR LSPNPWQEKR EIVSSEDAVTPSAVTPSAPS ASARPFIPVT 3501 DDLGAASIIA ETMTKTKEDV ESQNKAAGPE PQALDEFTSLLIADDTRVVV 3551 DLLKLSVCSR AGDRGRDVLS AVLSGMGTAY PQVADMLLEL CVTELEDVAT3601 DSQSGRLSSQ PVVVESSHPY TDDTSTSGTV KIPGAEGLRV EFDRQCSTER 3651RHDPLTVMDG VNRIVSVRSG REWSDWSSEL RIPGDELKWK FISDGSVNGW 3701 GWRFTVYPIMPAAGPKELLS DRCVLSCPSM DLVTCLLDFR LNLASNRSIV 3751 PRLAASLAAC AQLSALAASHRMWALQRLRK LLTTEFGQSI NINRLLGEND 3801 GETRALSFTG SALAALVKGL PEALQRQFEYEDPIVRGGKQ LLHSPFFKVL 3851 VALACDLELD TLPCCAETHK WAWFRRYCMA SRVAVALDKRTPLPRLFLDE 3901 VAKKIRELMA DSENMDVLHE SHDIFKREQD EQLVQWMNRR PDDWTLSAGG3951 SGTIYGWGHN HRGQLGGIEG AKVKVPTPCE ALATLRPVQL IGGEQTLFAV 4001TADGKLYATG YGAGGRLGIG GTESVSTPTL LESIQHVFIK KVAVNSGGKH 4051 CLALSSEGEVYSWGEAEDGK LGHGNRSPCD RPRVIESLRG IEVVDVAAGG 4101 AHSACVTAAG DLYTWGKGRYGRLGHSDSED QLKPKLVEAL QGHRVVDIAC 4151 GSGDAQTLCL TDDDTVWSWG DGDYGKLGRGGSDGCKVPMK IDSLTGLGVV 4201 KVECGSQFSV ALTKSGAVYT WGKGDYHRLG HGSDDHVRRPRQVQGLQGKK 4251 VIAIATGSLH CVCCTEDGEV YTWGDNDEGQ LGDGTTNAIQ RPRLVAALQG4301 KKVNRVACGS AHTLAWSTSK PASAGKLPAQ VPMEYNHLQE IPIIALRNRL 4351LLLHHLSELF CPCIPMFDLE GSLDETGLGP SVGFDTLRGI LISQGKEAAF 4401 RKVVQATMVRDRQHGPVVEL NRIQVKRSRS KGGLAGPDGT KSVFGQMCAK 4451 MSSFGPDSLL LPHRVWKVKFVGESVDDCGG GYSESIAEIC EELQNGLTPL 4501 LIVTPNGRDE SGANRDCYLL SPAARAPVHSSMFRFLGVLL GIAIRTGSPL 4551 SLNLAEPVWK QLAGMSLTIA DLSEVDKDFI PGLMYIRDNEATSEEFEAMS 4601 LPFTVPSASG QDIQLSSKHT HITLDNRAEY VRLAINYRLH EFDEQVAAVR4651 EGMARVVPVP LLSLFTGYEL ETMVCGSPDI PLHLLKSVAT YKGIEPSASL 4701IQWFWEVMES FSNTERSLFL RFVWGRTRLP RTIADFRGRD FVIQVLDKYN 4751 PPDHFLPESYTCFFLLKLPR YSCKQVLEEK LKYAIHFCKS IDTDDYARIA 4801 LTGEPAADDS SDDSDNEDVDSFASDSTQDY LTGH

E3 Ubiquitin-Protein Ligase HERC2-Derived Peptide HERC2

(SEQ ID NO: 12) KLAELPAAAQPSAEDSD

(7) Prothrombin-Derived Peptide THRB

For THRB shown as SEQ ID NO: 14, area values of cognitive impairment(AD, MCI, DLB and FTD) were significantly higher than NDC. (t-test,p<0.05) (see FIG. 10)

Thus, it was revealed that THRB shown as SEQ ID NO: 14 was useful todistinguish patient of cognitive impairment (AD, MCI, DLB and FTD) withsubjects not suffering from psychiatry disease (NDC). According to theanalysis by receiver operating characteristic (ROC) curve, CO3 wasclearly useful to distinguish AD and MCI with NDC. (See Table 1)

Intact Protein/Peptide

(SEQ ID NO: 13) 0001 ANTFLEEVRK GNLERECVEE TCSYEEAFEA LESSTATDVFWAKYTACETA 0051 RTPRDKLAAC LEGNCAEGLG TNYRGHVNIT RSGIECQLWR SRYPHKPEIN0101 STTHPGADLQ ENFCRNPDSS TTGPWCYTTD PTVRRQECSI PVCGQDQVTV 0151AMTPRSEGSS VNLSPPLEQC VPDRGQQYQG RLAVTTHGLP CLAWASAQAK 0201 ALSKHQDFNSAVQLVENFCR NPDGDEEGVW CYVAGKPGDF GYCDLNYCEE 0251 AVEEETGDGL DEDSDRAIEGRTATSEYQTF FNPRTFGSGE ADCGLRPLFE 0301 KKSLEDKTER ELLESYIDGR IVEGSDAEIGMSPWQVMLFR KSPQELLCGA 0351 SLISDRWVLT AAHCLLYPPW DKNFTENDLL VRIGKHSRTRYERNIEKISM 0401 LEKIYIHPRY NWRENLDRDI ALMKLKKPVA FSDYIHPVCL PDRETAASLL0451 QAGYKGRVTG WGNLKETWTA NVGKGQPSVL QVVNLPIVER PVCKDSTRIR 0501ITDNMFCAGY KPDEGKRGDA CEGDSGGPFV MKSPFNNRWY QMGIVSWGEG 0551 CDRDGKYGFYTHVFRLKKWI QKVIDQFGE

Prothrombin-Derived Peptide THRB

(SEQ ID NO: 14) TATSEYQTFFNPRTFGSGEAD

(8) Transthyretin-Derived Peptide TTHY

For TTHY shown as SEQ ID NO: 16, area values of cognitive impairment(AD, MCI, DLB and FTD) were significantly higher than NDC. (t-test,p<0.05) (see FIG. 11)

Thus, it was revealed that TTHY shown as SEQ ID NO: 16 was useful todistinguish patient of cognitive impairment (AD, MCI, DLB and FTD) withsubjects not suffering from psychiatry disease (NDC). According to theanalysis by receiver operating characteristic (ROC) curve, CO3 wasclearly useful to distinguish AD and MCI with NDC. (See Table 1)

Intact Protein/Peptide

(SEQ ID NO: 15) 0001 GPTGTGESKC PLMVKVLDAV RGSPAINVAV HVFRKAADDTWEPFASGKTS 0051 ESGELHGLTT EEEFVEGIYK VEIDTKSYWK ALGISPFHEH AEVVFTANDS0101 GPRRYTIAAL LSPYSYSTTA VVTNPKE

Transthyretin-Derived Peptide TTHY

(SEQ ID NO: 16) AVRGSPAINVAVHVFRKAAD

(9) Tumor Necrosis Factor Receptor Superfamily Member 16-Derived PeptideTNR16

For TNR16 shown as SEQ ID NO: 18, area values of cognitive impairment(AD, MCI, DLB and FTD) were significantly higher than NDC. (t-test,p<0.05) (see FIG. 12)

Thus, it was revealed that TNR16 shown as SEQ ID NO: 18 was useful todistinguish patient of cognitive impairment (AD, MCI, DLB and FTD) withsubjects not suffering from psychiatry disease (NDC). According to theanalysis by receiver operating characteristic (ROC) curve, CO3 wasclearly useful to distinguish AD and MCI with NDC. (See Table 1)

Intact Protein/Peptide

(SEQ ID NO: 17) 0001 KEACPTGLYT HSGECCKACN LGEGVAQPCG ANQTVCEPCLDSVTFSDVVS 0051 ATEPCKPCTE CVGLQSMSAP CVEADDAVCR CAYGYYQDET TGRCEACRVC0101 EAGSGLVFSC QDKQNTVCEE CPDGTYSDEA NHVDPCLPCT VCEDTERQLR 0151ECTRWADAEC EEIPGRWITR STPPEGSDST APSTQEPEAP PEQDLIASTV 0201 AGVVTTVMGSSQPVVTRGTT DNLIPVYCSI LAAVVVGLVA YIAFKRWNSC 0251 KQNKQGANSR PVNQTPPPEGEKLHSDSGIS VDSQSLHDQQ PHTQTASGQA 0301 LKGDGGLYSS LPPAKREEVE KLLNGSAGDTWRHLAGELGY QPEHIDSFTH 0351 EACPVRALLA SWATQDSATL DALLAALRRI QRADLVESLCSESTATSPV

Tumor Necrosis Factor Receptor Superfamily Member 16-Derived PeptideTNR16

(SEQ ID NO: 18) QTASGQALKGDGGLYS

(10) Complement C4-A-Derived Peptide CO4-1

For CO4-1 shown as SEQ ID NO: 20, area values of cognitive impairment(AD, MCI, DLB and FTD) were significantly higher than NDC. (t-test,p<0.05) (see FIG. 13)

Thus, it was revealed that CO4-1 shown as SEQ ID NO: 20 was useful todistinguish patient of cognitive impairment (AD, MCI, DLB and FTD) withsubjects not suffering from psychiatry disease (NDC). According to theanalysis by receiver operating characteristic (ROC) curve, CO3 wasclearly useful to distinguish AD and MCI with NDC. (See Table 1)

After Biosynthesis, Complement C4-A protein is divided into C4 betachain, Complement C4-A alpha chain and Complement C4 gamma chain byprocessing.

SEQ ID NO: 19 is amino acid sequence of intact Complement C4-A proteincontaining all of these processed peptides.

Intact Protein/Peptide

(SEQ ID NO: 19) 0001 KPRLLLFSPS VVHLGVPLSV GVQLQDVPRG QVVKGSVFLRNPSRNNVPCS 0051 PKVDFTLSSE RDFALLSLQV PLKDAKSCGL HQLLRGPEVQ LVAHSPWLKD0101 SLSRTTNIQG INLLFSSRRG HLFLQTDQPI YNPGQRVRYR VFALDQKMRP 0151STDTITVMVE NSHGLRVRKK EVYMPSSIFQ DDFVIPDISE PGTWKISARF 0201 SDGLESNSSTQFEVKKYVLP NFEVKITPGK PYILTVPGHL DEMQLDIQAR 0251 YIYGKPVQGV AYVRFGLLDEDGKKTFFRGL ESQTKLVNGQ SHISLSKAEF 0301 QDALEKLNMG ITDLQGLRLY VAAAIIESPGGEMEEAELTS WYFVSSPFSL 0351 DLSKTKRHLV PGAPFLLQAL VREMSGSPAS GIPVKVSATVSSPGSVPEVQ 0401 DIQQNTDGSG QVSIPIIIPQ TISELQLSVS AGSPHPAIAR LTVAAPPSGG0451 PGFLSIERPD SRPPRVGDTL NLNLRAVGSG ATFSHYYYMI LSRGQIVFMN 0501REPKRTLTSV SVFVDHHLAP SFYFVAFYYH GDHPVANSLR VDVQAGACEG 0551 KLELSVDGAKQYRNGESVKL HLETDSLALV ALGALDTALY AAGSKSHKPL 0601 NMGKVFEAMN SYDLGCGPGGGDSALQVFQA AGLAFSDGDQ WTLSRKRLSC 0651 PKEKTTRKKR NVNFQKAINE KLGQYASPTAKRCCQDGVTR LPMMRSCEQR 0701 AARVQQPDCR EPFLSCCQFA ESLRKKSRDK GQAGLQRALEILQEEDLIDE 0751 DDIPVRSFFP ENWLWRVETV DRFQILTLWL PDSLTTWEIH GLSLSKTKGL0801 CVATPVQLRV FREFHLHLRL PMSVRRFEQL ELRPVLYNYL DKNLTVSVHV 0851SPVEGLCLAG GGGLAQQVLV PAGSARPVAF SVVPTAAAAV SLKVVARGSF 0901 EFPVGDAVSKVLQIEKEGAI HREELVYELN PLDHRGRTLE IPGNSDPNMI 0951 PDGDFNSYVR VTASDPLDTLGSEGALSPGG VASLLRLPRG CGEQTMIYLA 1001 PTLAASRYLD KTEQWSTLPP ETKDHAVDLIQKGYMRIQQF RKADGSYAAW 1051 LSRDSSTWLT AFVLKVLSLA QEQVGGSPEK LQETSNWLLSQQQADGSFQD 1101 PCPVLDRSMQ GGLVGNDETV ALTAFVTIAL HHGLAVFQDE GAEPLKQRVE1151 ASISKANSFL GEKASAGLLG AHAAAITAYA LSLTKAPVDL LGVAHNNLMA 1201MAQETGDNLY WGSVTGSQSN AVSPTPAPRN PSDPMPQAPA LWIETTAYAL 1251 LHLLLHEGKAEMADQASAWL TRQGSFQGGF RSTQDTVIAL DALSAYWIAS 1301 HTTEERGLNVTLSSTGRNGF KSHALQLNNR QIRGLEEELQ FSLGSKINVK 1351 VGGNSKGTLK VLRTYNVLDMKNTTCQDLQI EVTVKGHVEY TMEANEDYED 1401 YEYDELPAKD DPDAPLQPVT PLQLFEGRRNRRRREAPKVV EEQESRVHYT 1451 VCIWRNGKVG LSGMAIADVT LLSGFHALRA DLEKLTSLSDRYVSHFETEG 1501 PHVLLYFDSV PTSRECVGFE AVQEVPVGLV QPASATLYDY YNPERRCSVF1551 YGAPSKSRLL ATLCSAEVCQ CAEGKCPRQR RALERGLQDE DGYRMKFACY 1601YPRVEYGFQV KVLREDSRAA FRLFETKITQ VLHFTKDVKA AANQMRNFLV 1651 RASCRLRLEPGKEYLIMGLD GATYDLEGHP QYLLDSNSWI EEMPSERLCR 1701 STRQRAACAQ LNDFLQEYGTQGCQV

Complement C4-Derived Peptide C04-1

(SEQ ID NO: 20) NGFKSHALQLNNRQIR

(11) Complement C4-B-Derived Peptide C04-1

From the results of MS/MS analysis and MASCOT database search, Asequence of CO4-1 peptide as shown SEQ ID NO: 20 is an amino acidsequence present in the part of topological region that is common toComplement C4-A protein (SEQ ID NO: 19) and Complement C4-B protein.After Biosynthesis, Complement C4-B protein is divided into C4 betachain, Complement C4-B alpha chain and Complement C4 gamma chain byprocessing.

SEQ ID NO: 21 is amino acid sequence of intact Complement C4-B proteincontaining all of these processed peptides.

Intact Protein/Peptide

(SEQ ID NO: 21) 0001 KPRLLLFSPS VVHLGVPLSV GVQLQDVPRG QVVKGSVFLRNPSRNNVPCS 0051 PKVDFTLSSE RDFALLSLQV PLKDAKSCGL HQLLRGPEVQ LVAHSPWLKD0101 SLSRTTNIQG INLLFSSRRG HLFLQTDQPI YNPGQRVRYR VFALDQKMRP 0151STDTITVMVE NSHGLRVRKK EVYMPSSIFQ DDFVIPDISE PGTWKISARF 0201 SDGLESNSSTQFEVKKYVLP NFEVKITPGK PYILTVPGHL DEMQLDIQAR 0251 YIYGKPVQGV AYVRFGLLDEDGKKTFFRGL ESQTKLVNGQ SHISLSKAEF 0301 QDALEKLNMG ITDLQGLRLY VAAAIIESPGGEMEEAELTS WYFVSSPFSL 0351 DLSKTKRHLV PGAPFLLQAL VREMSGSPAS GIPVKVSATVSSPGSVPEVQ 0401 DIQQNTDGSG QVSIPIIIPQ TISELQLSVS AGSPHPAIAR LTVAAPPSGG0451 PGFLSIERPD SRPPRVGDTL NLNLRAVGSG ATFSHYYYMI LSRGQIVFMN 0501REPKRTLTSV SVFVDHHLAP SFYFVAFYYH GDHPVANSLR VDVQAGACEG 0551 KLELSVDGAKQYRNGESVKL HLETDSLALV ALGALDTALY AAGSKSHKPL 0601 NMGKVFEAMN SYDLGCGPGGGDSALQVFQA AGLAFSDGDQ WTLSRKRLSC 0651 PKEKTTRKKR NVNFQKAINE KLGQYASPTAKRCCQDGVTR LPMMRSCEQR 0701 AARVQQPDCR EPFLSCCQFA ESLRKKSRDK GQAGLQRALEILQEEDLIDE 0751 DDIPVRSFFP ENWLWRVETV DRFQILTLWL PDSLTTWEIH GLSLSKTKGL0801 CVATPVQLRV FREFHLHLRL PMSVRRFEQL ELRPVLYNYL DKNLTVSVHV 0851SPVEGLCLAG GGGLAQQVLV PAGSARPVAF SVVPTAAAAV SLKVVARGSF 0901 EFPVGDAVSKVLQIEKEGAI HREELVYELN PLDHRGRTLE IPGNSDPNMI 0951 PDGDFNSYVR VTASDPLDTLGSEGALSPGG VASLLRLPRG CGEQTMIYLA 1001 PTLAASRYLD KTEQWSTLPP ETKDHAVDLIQKGYMRIQQF RKADGSYAAW 1051 LSRDSSTWLT AFVLKVLSLA QEQVGGSPEK LQETSNWLLSQQQADGSFQD 1101 LSPVIHRSMQ GGLVGNDETV ALTAFVTIAL HHGLAVFQDE GAEPLKQRVE1151 ASISKANSFL GEKASAGLLG AHAAAITAYA LSLTKAPVDL LGVAHNNLMA 1201MAQETGDNLY WGSVTGSQSN AVSPTPAPRN PSDPMPQAPA LWIETTAYAL 1251 LHLLLHEGKAEMADQASAWL TRQGSFQGGF RSTQDTVIAL DALSAYWIAS 1301 HTTEERGLNVTLSSTGRNGF KSHALQLNNR QIRGLEEELQ FSLGSKINVK 1351 VGGNSKGTLK VLRTYNVLDMKNTTCQDLQI EVTVKGHVEY TMEANEDYED 1401 YEYDELPAKD DPDAPLQPVT PLQLFEGRRNRRRREAPKVV EEQESRVHYT 1451 VCIWRNGKVG LSGMAIADVT LLSGFHALRA DLEKLTSLSDRYVSHFETEG 1501 PHVLLYFDSV PTSRECVGFE AVQEVPVGLV QPASATLYDY YNPERRCSVF1551 YGAPSKSRLL ATLCSAEVCQ CAEGKCPRQR RALERGLQDE DGYRMKFACY 1601YPRVEYGFQV KVLREDSRAA FRLFETKITQ VLHFTKDVKA AANQMRNFLV 1651 RASCRLRLEPGKEYLIMGLD GATYDLEGHP QYLLDSNSWI EEMPSERLCR 1701 STRQRAACAQ LNDFLQEYGTQGCQVJust in case, following shows the sequence of CO4-1.

ComplementC4-Derived Peptide CO4-1

(SEQ ID NO: 20) NGFKSHALQLNNRQIR

(12) Complement C4-A-Derived Peptide CO4-2

For CO4-2 shown as SEQ ID NO: 22, area values of cognitive impairment(AD, MCI, DLB and FTD) were significantly higher than NDC. (t-test,p<0.05) (see FIG. 14)

Thus, it was revealed that CO4-2 shown as SEQ ID NO: 22 was useful todistinguish patient of cognitive impairment (AD, MCI, DLB and FTD) withsubjects not suffering from psychiatry disease (NDC). According to theanalysis by receiver operating characteristic (ROC) curve, CO3 wasclearly useful to distinguish AD and MCI with NDC. (See Table 1) AfterBiosynthesis, Complement C4-A protein is divided into C4 beta chain,Complement C4-A alpha chain and Complement C4 gamma chain by processing.

SEQ ID NO: 19 is amino acid sequence of intact Complement C4-A proteincontaining all of these processed peptides.

Intact Protein/Peptide

(SEQ ID NO: 19) 0001 KPRLLLFSPS VVHLGVPLSV GVQLQDVPRG QVVKGSVFLRNPSRNNVPCS 0051 PKVDFTLSSE RDFALLSLQV PLKDAKSCGL HQLLRGPEVQ LVAHSPWLKD0101 SLSRTTNIQG INLLFSSRRG HLFLQTDQPI YNPGQRVRYR VFALDQKMRP 0151STDTITVMVE NSHGLRVRKK EVYMPSSIFQ DDFVIPDISE PGTWKISARF 0201 SDGLESNSSTQFEVKKYVLP NFEVKITPGK PYILTVPGHL DEMQLDIQAR 0251 YIYGKPVQGV AYVRFGLLDEDGKKTFFRGL ESQTKLVNGQ SHISLSKAEF 0301 QDALEKLNMG ITDLQGLRLY VAAAIIESPGGEMEEAELTS WYFVSSPFSL 0351 DLSKTKRHLV PGAPFLLQAL VREMSGSPAS GIPVKVSATVSSPGSVPEVQ 0401 DIQQNTDGSG QVSIPIIIPQ TISELQLSVS AGSPHPAIAR LTVAAPPSGG0451 PGFLSIERPD SRPPRVGDTL NLNLRAVGSG ATFSHYYYMI LSRGQIVFMN 0501REPKRTLTSV SVFVDHHLAP SFYFVAFYYH GDHPVANSLR VDVQAGACEG 0551 KLELSVDGAKQYRNGESVKL HLETDSLALV ALGALDTALY AAGSKSHKPL 0601 NMGKVFEAMN SYDLGCGPGGGDSALQVFQA AGLAFSDGDQ WTLSRKRLSC 0651 PKEKTTRKKR NVNFQKAINE KLGQYASPTAKRCCQDGVTR LPMMRSCEQR 0701 AARVQQPDCR EPFLSCCQFA ESLRKKSRDK GQAGLQRALEILQEEDLIDE 0751 DDIPVRSFFP ENWLWRVETV DRFQILTLWL PDSLTTWEIH GLSLSKTKGL0801 CVATPVQLRV FREFHLHLRL PMSVRRFEQL ELRPVLYNYL DKNLTVSVHV 0851SPVEGLCLAG GGGLAQQVLV PAGSARPVAF SVVPTAAAAV SLKVVARGSF 0901 EFPVGDAVSKVLQIEKEGAI HREELVYELN PLDHRGRTLE IPGNSDPNMI 0951 PDGDFNSYVR VTASDPLDTLGSEGALSPGG VASLLRLPRG CGEQTMIYLA 1001 PTLAASRYLD KTEQWSTLPP ETKDHAVDLIQKGYMRIQQF RKADGSYAAW 1051 LSRDSSTWLT AFVLKVLSLA QEQVGGSPEK LQETSNWLLSQQQADGSFQD 1101 PCPVLDRSMQ GGLVGNDETV ALTAFVTIAL HHGLAVFQDE GAEPLKQRVE1151 ASISKANSFL GEKASAGLLG AHAAAITAYA LSLTKAPVDL LGVAHNNLMA 1201MAQETGDNLY WGSVTGSQSN AVSPTPAPRN PSDPMPQAPA LWIETTAYAL 1251 LHLLLHEGKAEMADQASAWL TRQGSFQGGF RSTQDTVIAL DALSAYWIAS 1301 HTTEERGLNV TLSSTGRNGFKSHALQLNNR QIRGLEEELQ FSLGSKINVK 1351 VGGNSKGTLK VLRTYNVLDM KNTTCQDLQIEVTVKGHVEY TMEANEDYED 1401 YEYDELPAKD DPDAPLQPVT PLQLFEGRRN RRRREAPKVVEEQESRVHYT 1451 VCIWRNGKVG LSGMAIADVT LLSGFHALRA DLEKLTSLSD RYVSHFETEG1501 PHVLLYFDSV PTSRECVGFE AVQEVPVGLV QPASATLYDY YNPERRCSVF 1551YGAPSKSRLL ATLCSAEVCQ CAEGKCPRQR RALERGLQDE DGYRMKFACY 1601 YPRVEYGFQVKVLREDSRAA FRLFETKITQ VLHFTKDVKA AANQMRNFLV 1651 RASCRLRLEP GKEYLIMGLDGATYDLEGHP QYLLDSNSWI EEMPSERLCR 1701 STRQRAACAQ LNDFLQEYGT QGCQV

Complement C4-Derived Peptide CO4-2

(SEQ ID NO: 22) APLQPVTPLQLFEGRRN

(13) Complement C4-B-Derived Peptide CO4-2

From the results of MS/MS analysis and MASCOT database search, Asequence of CO4-2 peptide as shown SEQ ID NO: 22 is an amino acidsequence present in the part of topological region that is common toComplement C4-A protein (SEQ ID NO: 19) and Complement C4-B protein.After Biosynthesis, Complement C4-B protein is divided into C4 betachain, Complement C4-B alpha chain and Complement C4 gamma chain byprocessing. SEQ ID NO: 21 is amino acid sequence of intact ComplementC4-B protein containing all of these processed peptides.

Intact Protein/Peptide

(SEQ ID NO: 21) 0001 KPRLLLFSPS VVHLGVPLSV GVQLQDVPRG QVVKGSVFLRNPSRNNVPCS 0051 PKVDFTLSSE RDFALLSLQV PLKDAKSCGL HQLLRGPEVQ LVAHSPWLKD0101 SLSRTTNIQG INLLFSSRRG HLFLQTDQPI YNPGQRVRYR VFALDQKMRP 0151STDTITVMVE NSHGLRVRKK EVYMPSSIFQ DDFVIPDISE PGTWKISARF 0201 SDGLESNSSTQFEVKKYVLP NFEVKITPGK PYILTVPGHL DEMQLDIQAR 0251 YIYGKPVQGV AYVRFGLLDEDGKKTFFRGL ESQTKLVNGQ SHISLSKAEF 0301 QDALEKLNMG ITDLQGLRLY VAAAIIESPGGEMEEAELTS WYFVSSPFSL 0351 DLSKTKRHLV PGAPFLLQAL VREMSGSPAS GIPVKVSATVSSPGSVPEVQ 0401 DIQQNTDGSG QVSIPIIIPQ TISELQLSVS AGSPHPAIAR LTVAAPPSGG0451 PGFLSIERPD SRPPRVGDTL NLNLRAVGSG ATFSHYYYMI LSRGQIVFMN 0501REPKRTLTSV SVFVDHHLAP SFYFVAFYYH GDHPVANSLR VDVQAGACEG 0551 KLELSVDGAKQYRNGESVKL HLETDSLALV ALGALDTALY AAGSKSHKPL 0601 NMGKVFEAMN SYDLGCGPGGGDSALQVFQA AGLAFSDGDQ WTLSRKRLSC 0651 PKEKTTRKKR NVNFQKAINE KLGQYASPTAKRCCQDGVTR LPMMRSCEQR 0701 AARVQQPDCR EPFLSCCQFA ESLRKKSRDK GQAGLQRALEILQEEDLIDE 0751 DDIPVRSFFP ENWLWRVETV DRFQILTLWL PDSLTTWEIH GLSLSKTKGL0801 CVATPVQLRV FREFHLHLRL PMSVRRFEQL ELRPVLYNYL DKNLTVSVHV 0851SPVEGLCLAG GGGLAQQVLV PAGSARPVAF SVVPTAAAAV SLKVVARGSF 0901 EFPVGDAVSKVLQIEKEGAI HREELVYELN PLDHRGRTLE IPGNSDPNMI 0951 PDGDFNSYVR VTASDPLDTLGSEGALSPGG VASLLRLPRG CGEQTMIYLA 1001 PTLAASRYLD KTEQWSTLPP ETKDHAVDLIQKGYMRIQQF RKADGSYAAW 1051 LSRDSSTWLT AFVLKVLSLA QEQVGGSPEK LQETSNWLLSQQQADGSFQD 1101 LSPVIHRSMQ GGLVGNDETV ALTAFVTIAL HHGLAVFQDE GAEPLKQRVE1151 ASISKANSFL GEKASAGLLG AHAAAITAYA LSLTKAPVDL LGVAHNNLMA 1201MAQETGDNLY WGSVTGSQSN AVSPTPAPRN PSDPMPQAPA LWIETTAYAL 1251 LHLLLHEGKAEMADQASAWL TRQGSFQGGF RSTQDTVIAL DALSAYWIAS 1301 HTTEERGLNV TLSSTGRNGFKSHALQLNNR QIRGLEEELQ FSLGSKINVK 1351 VGGNSKGTLK VLRTYNVLDM KNTTCQDLQIEVTVKGHVEY TMEANEDYED 1401 YEYDELPAKD DPDAPLQPVT PLQLFEGRRN RRRREAPKVVEEQESRVHYT 1451 VCIWRNGKVG LSGMAIADVT LLSGFHALRA DLEKLTSLSD RYVSHFETEG1501 PHVLLYFDSV PTSRECVGFE AVQEVPVGLV QPASATLYDY YNPERRCSVF 1551YGAPSKSRLL ATLCSAEVCQ CAEGKCPRQR RALERGLQDE DGYRMKFACY 1601 YPRVEYGFQVKVLREDSRAA FRLFETKITQ VLHFTKDVKA AANQMRNFLV 1651 RASCRLRLEP GKEYLIMGLDGATYDLEGHP QYLLDSNSWI EEMPSERLCR 1701 STRQRAACAQ LNDFLQEYGT QGCQV

Just in case, following shows the sequence of CO4-2.

Complement C4-Derived Peptide CO4-2

(SEQ ID NO: 22) APLQPVTPLQLFEGRRN

(14) Fibrinogen Alpha Chain (Isoform 1)-Derived Peptide FIBA-1

For FIBA-1 shown as SEQ ID NO: 24, area values of cognitive impairment(AD, MCI, DLB and FTD) were significantly higher than NDC. (t-test,p<0.05) (see FIG. 15)

Thus, it was revealed that FIBA-1 shown as SEQ ID NO: 24 was useful todistinguish patient of cognitive impairment (AD, MCI, DLB and FTD) withsubjects not suffering from psychiatry disease (NDC). According to theanalysis by receiver operating characteristic (ROC) curve, CO3 wasclearly useful to distinguish AD and MCI with NDC.

(See Table 1) Intact Protein/Peptide

(SEQ ID NO: 23) 0001 GPRVVERHQS ACKDSDWPFC SDEDWNYKCP SGCRMKGLIDEVNQDFTNRI 0051 NKLKNSLFEY QKNNKDSHSL TTNIMEILRG DFSSANNRDN TYNRVSEDLR0101 SRIEVLKRKV IEKVQHIQLL QKNVRAQLVD MKRLEVDIDI KIRSCRGSCS 0151RALAREVDLK DYEDQQKQLE QVIAKDLLPS RDRQHLPLIK MKPVPDLVPG 0201 NFKSQLQKVPPEWKALTDMP QMRMELERPG GNEITRGGST SYGTGSETES 0251 PRNPSSAGSW NSGSSGPGSTGNRNPGSSGT GGTATWKPGS SGPGSTGSWN 0301 SGSSGTGSTG NQNPGSPRPG STGTWNPGSSERGSAGHWTS ESSVSGSTGQ 0351 WHSESGSFRP DSPGSGNARP NNPDWGTFEE VSGNVSPGTRREYHTEKLVT 0401 SKGDKELRTG KEKVTSGSTT TTRRSCSKTV TKTVIGPDGH KEVTKEVVTS0451 EDGSDCPEAM DLGTLSGIGT LDGFRHRHPD EAAFFDTAST GKTFPGFFSP 0501MLGEFVSETE SRGSESGIFT NTKESSSHHP GIAEFPSRGK SSSYSKQFTS 0551STSYNRGDST FESKSYKMAD EAGSEADHEG THSTKRGHAK SRPVRDCDDV 0601 LQTHPSGTQSGIFNIKLPGS SKIFSVYCDQ ETSLGGWLLI QQRMDGSLNF 0651 NRTWQDYKRG FGSLNDEGEGEFWLGNDYLH LLTQRGSVLR VELEDWAGNE 0701 AYAEYHFRVG SEAEGYALQV SSYEGTAGDALIEGSVEEGA EYTSHNNMQF 0751 STFDRDADQW EENCAEVYGG GWWYNNCQAA NLNGIYYPGGSYDPRNNSPY 0801 EIENGVVWVS FRGADYSLRA VRMKIRPLVT Q

Fibrinogen Alpha Chain-Derived Peptide FIBA-1

(SEQ ID NO: 24) SSSYSKQFTSSTSYNRGDSTFES

(15) Fibrinogen Alpha Chain (Isoform 2)-Derived Peptide FIBA-1

From the results of MS/MS analysis and MASCOT database search, Asequence of FIBA-1 peptide as shown SEQ ID NO: 24 is an amino acidsequence present in the part of topological region that is common toFibrinogen alpha chain (isoform 1) (SEQ ID NO: 23) and Fibrinogen alphachain (isoform 2). Followings, as SEQ ID NO: 25, an amino acid sequenceof intact protein of Fibrinogen alpha chain (isoform 2) were shown.

Intact Protein/Peptide

(SEQ ID NO: 25) 0001 GPRVVERHQS ACKDSDWPFC SDEDWNYKCP SGCRMKGLIDEVNQDFTNRI 0051 NKLKNSLFEY QKNNKDSHSL TTNIMEILRG DFSSANNRDN TYNRVSEDLR0101 SRIEVLKRKV IEKVQHIQLL QKNVRAQLVD MKRLEVDIDI KIRSCRGSCS 0151RALAREVDLK DYEDQQKQLE QVIAKDLLPS RDRQHLPLIK MKPVPDLVPG 0201 NFKSQLQKVPPEWKALTDMP QMRMELERPG GNEITRGGST SYGTGSETES 0251 PRNPSSAGSW NSGSSGPGSTGNRNPGSSGT GGTATWKPGS SGPGSTGSWN 0301 SGSSGTGSTG NQNPGSPRPG STGTWNPGSSERGSAGHWTS ESSVSGSTGQ 0351 WHSESGSFRP DSPGSGNARP NNPDWGTFEE VSGNVSPGTRREYHTEKLVT 0401 SKGDKELRTG KEKVTSGSTT TTRRSCSKTV TKTVIGPDGH KEVTKEVVTS0451 EDGSDCPEAM DLGTLSGIGT LDGFRHRHPD EAAFFDTAST GKTFPGFFSP 0501MLGEFVSETE SRGSESGIFT NTKESSSHHP GIAEFPSRGK SSSYSKQFTS 0551STSYNRGDST FESKSYKMAD EAGSEADHEG THSTKRGHAK SRPVRGIHTS 0601 PLGKPSLSP

Just in case, following shows the sequence of FIBA-1.

Fibrinogen Alpha Chain-Derived Peptide FIBA-1

(SEQ ID NO: 24) SSSYSKQFTSSTSYNRGDSTFES

(16) Fibrinogen Alpha Chain (Isoform 1)-Derived Peptide FIBA-2

For FIBA-2 shown as SEQ ID NO: 26, area values of cognitive impairment(AD, MCI, DLB and FTD) were significantly higher than NDC. (t-test,p<0.05) (see FIG. 16)

Thus, it was revealed that FIBA-2 shown as SEQ ID NO: 26 was useful todistinguish patient of cognitive impairment (AD, MCI, DLB and FTD) withsubjects not suffering from psychiatry disease (NDC). According to theanalysis by receiver operating characteristic (ROC) curve, CO3 wasclearly useful to distinguish AD and MCI with NDC.

(See Table 1) Intact Protein/Peptide

(SEQ ID NO: 23) 0001 GPRVVERHQS ACKDSDWPFC SDEDWNYKCP SGCRMKGLIDEVNQDFTNRI 0051 NKLKNSLFEY QKNNKDSHSL TTNIMEILRG DFSSANNRDN TYNRVSEDLR0101 SRIEVLKRKV IEKVQHIQLL QKNVRAQLVD MKRLEVDIDI KIRSCRGSCS 0151RALAREVDLK DYEDQQKQLE QVIAKDLLPS RDRQHLPLIK MKPVPDLVPG 0201 NFKSQLQKVPPEWKALTDMP QMRMELERPG GNEITRGGST SYGTGSETES 0251 PRNPSSAGSW NSGSSGPGSTGNRNPGSSGT GGTATWKPGS SGPGSTGSWN 0301 SGSSGTGSTG NQNPGSPRPG STGTWNPGSSERGSAGHWTS ESSVSGSTGQ 0351 WHSESGSFRP DSPGSGNARP NNPDWGTFEE VSGNVSPGTRREYHTEKLVT 0401 SKGDKELRTG KEKVTSGSTT TTRRSCSKTV TKTVIGPDGH KEVTKEVVTS0451 EDGSDCPEAM DLGTLSGIGT LDGFRHRHPD EAAFFDTAST GKTFPGFFSP 0501MLGEFVSETE SRGSESGIFT NTKESSSHHP GIAEFPSRGK SSSYSKQFTS 0551STSYNRGDST FESKSYKMAD EAGSEADHEG THSTKRGHAK SRPVRDCDDV 0601 LQTHPSGTQSGIFNIKLPGS SKIFSVYCDQ ETSLGGWLLI QQRMDGSLNF 0651 NRTWQDYKRG FGSLNDEGEGEFWLGNDYLH LLTQRGSVLR VELEDWAGNE 0701 AYAEYHFRVG SEAEGYALQV SSYEGTAGDALIEGSVEEGA EYTSHNNMQF 0751 STFDRDADQW EENCAEVYGG GWWYNNCQAA NLNGIYYPGGSYDPRNNSPY 0801 EIENGVVWVS FRGADYSLRA VRMKIRPLVT Q

Fibrinogen Alpha Chain-Derived Peptide FIBA-2

(SEQ ID NO: 26) SSSYSKQFTSSTSYNRGDSTFESKS

(17) Fibrinogen Alpha Chain (Isoform 2)-Derived Peptide FIBA-2

From the results of MS/MS analysis and MASCOT database search, Asequence of FIBA-2 peptide as shown SEQ ID NO: 26 is an amino acidsequence present in the part of topological region that is common toFibrinogen alpha chain (isoform 1) (SEQ ID NO: 23) and Fibrinogen alphachain (isoform 2). Followings, as SEQ ID NO: 25, an amino acid sequenceof intact protein of Fibrinogen alpha chain (isoform 2) were shown.

Intact Protein/Peptide

(SEQ ID NO: 25) 0001 GPRVVERHQS ACKDSDWPFC SDEDWNYKCP SGCRMKGLIDEVNQDFTNRI 0051 NKLKNSLFEY QKNNKDSHSL YFNIMEILRG DFSSANNRDN TYNRVSEDLR0101 SRIEVLKRKV IEKVQHIQLL QKNVRAQLVD MKRLEVDIDI KIRSCRGSCS 0151RALAREVDLK DYEDQQKQLE QVIAKDLLPS RDRQHLPLIK MKPVPDLVPG 0201 NFKSQLQKVPPEWKALTDMP QMRMELERPG GNEITRGGST SYGTGSETES 0251 PRNPSSAGSW NSGSSGPGSTGNRNPGSSGT GGTATWKPGS SGPGSTGSWN 0301 SGSSGTGSTG NQNPGSPRPG STGTWNPGSSERGSAGHWTS ESSVSGSTGQ 0351 WHSESGSFRP DSPGSGNARP NNPDWGTFEE VSGNVSPGTRREYHTEKLVT 0401 SKGDKELRTG KEKVTSGSTT TTRRSCSKTV TKTVIGPDGH KEVTKEVVTS0451 EDGSDCPEAM DLGTLSGIGT LDGFRHRHPD EAAFFDTAST GKTFPGFFSP 0501MLGEFVSETE SRGSESGIFT NTKESSSHHP GIAEFPSRGK SSSYSKQFTS 0551STSYNRGDST FESKSYKMAD EAGSEADHEG THSTKRGHAK SRPVRGIHTS 0601 PLGKPSLSP

Just in case, following shows the sequence of FIBA-2.

Fibrinogen Alpha Chain-Derived Peptide FIBA-2

(SEQ ID NO: 26) SSSYSKQFTSSTSYNRGDSTFESKS

(18) Fibrinogen Alpha Chain (Isoform 1)-Derived Peptide FIBA-3

For FIBA-3 shown as SEQ ID NO: 27, area values of cognitive impairment(AD, MCI, DLB and FTD) were significantly higher than NDC. (t-test,p<0.05) (see FIG. 17)

Thus, it was revealed that FIBA-3 shown as SEQ ID NO: 27 was useful todistinguish patient of cognitive impairment (AD, MCI, DLB and FTD) withsubjects not suffering from psychiatry disease (NDC). According to theanalysis by receiver operating characteristic (ROC) curve, CO3 wasclearly useful to distinguish AD and MCI with NDC. (See Table 1)

Intact Protein/Peptide

(SEQ ID NO: 23) 0001 GPRVVERHQS ACKDSDWPFC SDEDWNYKCP SGCRMKGLIDEVNQDFTNRI 0051 NKLKNSLFEY QKNNKDSHSL TTNIMEILRG DFSSANNRDN TYNRVSEDLR0101 SRIEVLKRKV IEKVQHIQLL QKNVRAQLVD MKRLEVDIDI KIRSCRGSCS 0151RALAREVDLK DYEDQQKQLE QVIAKDLLPS RDRQHLPLIK MKPVPDLVPG 0201 NFKSQLQKVPPEWKALTDMP QMRMELERPG GNEITRGGST SYGTGSETES 0251 PRNPSSAGSW NSGSSGPGSTGNRNPGSSGT GGTATWKPGS SGPGSTGSWN 0301 SGSSGTGSTG NQNPGSPRPG STGTWNPGSSERGSAGHWTS ESSVSGSTGQ 0351 WHSESGSFRP DSPGSGNARP NNPDWGTFEE VSGNVSPGTRREYHTEKLVT 0401 SKGDKELRTG KEKVTSGSTT TTRRSCSKTV TKTVIGPDGH KEVTKEVVTS0451 EDGSDCPEAM DLGTLSGIGT LDGFRHRHPD EAAFFDTAST GKTFPGFFSP 0501MLGEFVSETE SRGSESGIFT NTKESSSHHP GIAEFPSRGK SSSYSKQFTS 0551STSYNRGDST FESKSYKMAD EAGSEADHEG THSTKRGHAK SRPVRDCDDV 0601 LQTHPSGTQSGIFNIKLPGS SKIFSVYCDQ ETSLGGWLLI QQRMDGSLNF 0651 NRTWQDYKRG FGSLNDEGEGEFWLGNDYLH LLTQRGSVLR VELEDWAGNE 0701 AYAEYHFRVG SEAEGYALQV SSYEGTAGDALIEGSVEEGA EYTSHNNMQF 0751 STFDRDADQW EENCAEVYGG GWWYNNCQAA NLNGIYYPGGSYDPRNNSPY 0801 EIENGVVWVS FRGADYSLRA VRMKIRPLVT Q

Fibrinogen Alpha Chain-Derived Peptide FIBA-3

(SEQ ID NO: 27) SSSYSKQFTSSTSYNRGDSTFESKSY

(19) Fibrinogen Alpha Chain (Isoform 2)-Derived Peptide FIBA-3

From the results of MS/MS analysis and MASCOT database search, Asequence of FIBA-3 peptide as shown SEQ ID NO: 27 is an amino acidsequence present in the part of topological region that is common toFibrinogen alpha chain (isoform 1) (SEQ ID NO: 23) and Fibrinogen alphachain (isoform 2). Followings, as SEQ ID NO: 25, an amino acid sequenceof intact protein of Fibrinogen alpha chain (isoform 2) were shown.

Intact Protein/Peptide

(SEQ ID NO: 25) 0001 GPRVVERHQS ACKDSDWPFC SDEDWNYKCP SGCRMKGLIDEVNQDFTNRI 0051 NKLKNSLFEY QKNNKDSHSL TTNIMEILRG DFSSANNRDN TYNRVSEDLR0101 SRIEVLKRKV IEKVQHIQLL QKNVRAQLVD MKRLEVDIDI KIRSCRGSCS 0151RALAREVDLK DYEDQQKQLE QVIAKDLLPS RDRQHLPLIK MKPVPDLVPG 0201 NFKSQLQKVPPEWKALTDMP QMRMELERPG GNEITRGGST SYGTGSETES 0251 PRNPSSAGSW NSGSSGPGSTGNRNPGSSGT GGTATWKPGS SGPGSTGSWN 0301 SGSSGTGSTG NQNPGSPRPG STGTWNPGSSERGSAGHWTS ESSVSGSTGQ 0351 WHSESGSFRP DSPGSGNARP NNPDWGTFEE VSGNVSPGTRREYHTEKLVT 0401 SKGDKELRTG KEKVTSGSTT TTRRSCSKTV TKTVIGPDGH KEVTKEVVTS0451 EDGSDCPEAM DLGTLSGIGT LDGFRHRHPD EAAFFDTAST GKTFPGFFSP 0501MLGEFVSETE SRGSESGIFT NTKESSSHHP GIAEFPSRGK SSSYSKQFTS 0551STSYNRGDST FESKSYKMAD EAGSEADHEG THSTKRGHAK SRPVRGIHTS 0601 PLGKPSLSP

Just in case, following shows the sequence of FIBA-3.

Fibrinogen Alpha Chain-Derived Peptide FIBA-3

(SEQ ID NO: 27) SSSYSKQFTSSTSYNRGDSTFESKSY

TABLE 1 Marker Peptide Sequence Sequence AD vs. NDC MCI vs. NDC No. nameAUC value AUC value 2 CO3 0.88 0.83 4 AP2C 0.78 0.70 6 SYN3 0.77 0.77 8OXYR 0.81 0.77 10 ITH5L 0.79 0.70 12 HERC2 0.76 0.73 14 THRB 0.85 0.7916 TTHY 0.73 0.69 18 TNR16 0.75 0.74 20 CO4-1 0.73 0.67 22 CO4-2 0.760.74 24 FIBA-1 0.77 0.64 26 FIBA-2 0.74 0.61 27 FIBA-3 0.80 0.64

Table 1 shows AUC values obtained by the analysis by receiver operatingcharacteristic (ROC) curve in the detection of cognitive impairment ofeach marker peptides.

Using these marker peptides in singly or in combination, using orwithout using liquid chromatography and/or any other suitable separationmethods, directly measuring the abundance in serum using other methodssuch as mass spectrometry or immunological methods or enzymatic methods,on the diagnosis, it is possible to distinguish between non-psychiatrydisease subjects including normal healthy subjects and subjects ofcognitive impairment like AD, MCI, DLB and FTD.

INDUSTRIAL APPLICABILITY

By using the biomarkers disclosed in the present invention, mildcognitive impairment and cognitive impairment including Alzheimerdisease can be detected. This invention is applicable to the field ofmedical diagnostics including diagnostic reagent.

SEQUENCE LIST

-   10P01009 Sequence.txt

1. A method of detecting Complement C3-derived peptide CO3 in a patient, said method comprising: a. obtaining a biological material from a human patient; and b. detecting presence or an amount of the Complement C3-derived peptide CO3 consisting of amino acid sequence of SEQ ID NO: 2 in the biological material by contacting the biological material with an antibody or aptamer that specifically binds to the peptide CO3 and detecting binding between the Complement C3-derived peptide CO3 and the antibody or aptamer.
 2. The method of claim 1, wherein the biological material is serum, blood, plasma, cerebrospinal fluid, or urine.
 3. A method of diagnosing and treating cognitive impairment in a patient, said method comprising: a. obtaining a biological material from a human patient; b. detecting an amount of Complement C3-derived peptide CO3 consisting of amino acid sequence of SEQ ID NO: 2 in the biological material by contacting the biological material with an antibody or aptamer that specifically binds to the peptide CO3 and detecting binding between the peptide CO3 and the antibody or aptamer; c. diagnosing the patient with cognitive impairment when a higher amount of the Complement C3-derived peptide CO3 in the biological material is detected by comparing the amount of the Complement C3-derived peptide CO3 in the patient with an amount of the Complement C3-derived peptide CO3 in a biological material obtained from a non-psychiatry disease subject; and d. administering an effective amount of an anti-acetylcholine esterase inhibitor to the diagnosed patient.
 4. The method of claim 3, wherein the biological material is serum, blood, plasma, cerebrospinal fluid, or urine.
 5. The method of claim 3, wherein the cognitive impairment includes Alzheimer's dementia, mild cognitive impairment, Dementia with Lewy bodies, and frontotemporal dementia.
 6. The method of claim 3, wherein the anti-acetylcholine esterase inhibitor is Donepezil-hydrochloride. 